H pylori antigens

ABSTRACT

The present invention discloses isolated nucleic acid molecules encoding a hyperimmune serum reactive antigen or a fragment thereof as well as hyperimmune serum reactive antigens or fragments thereof from  H. pylori , methods for isolating such antigens and specific uses thereof.

The present invention relates to isolated nucleic acid molecules, whichencode antigens for Helicobacter pylori, which are suitable for use inpreparation of pharmaceutical medicaments for the prevention andtreatment of bacterial infections caused by Helicobacter pylori.

Helicobacter pylori is a Gram-negative, microaerobic, spiral andflagellated bacterium representing the most prevalent human pathogenwith nearly half the globe's population infected. Infection most likelyoccurs in early childhood (<age 10) in most cases and the pathogenspecifically colonizes the stomach where it becomes a resident.Colonization lasts for years or even decades but it can persist forlife, yet about 70-80% of colonized individuals remain asymptomatic andnever develop disease. It is now clear that the prolonged interaction ofH. pylori with gastric epithelia is a complex and dynamic process, whichleads to chronic acute inflammation of the gastric mucosa and to thedevelopment of peptic ulcer disease in 10-20% of the cases. Nearly allduodenal ulcers are caused by H. pylori and, in the stomach ulcers candevelop into gastric adenocarcinoma with a frequency of 0.1-4%. Thesignificance of this number is that gastric cancer is the second mostcommon fatal malignancy after lung cancer and within twenty years, it ispredicted to be the 8th leading cause of death of any origin worldwide.H. pylori infection is also associated with about 90% ofmucosa-associated lymphoid tissue lymphomas (MALT). Patients with H.pylori infection develop high titers of primarily IgG and IgA antibodiesbut their role in the immune response against the bacterium is notknown. Presence of the bacteria within the mucosal epithelium isassociated with massive neutrophil infiltration. Considerable evidenceexists demonstrating that the H. pylori-induced Th1-biased CD4⁺ T cellresponse with prominent IFN-γ production might be a strong contributingfactor in the outcome of the local immune reaction linked to tissuedamage.

H. pylori is inherently equipped with an array of extremely potentfactors and mechanisms that enable the pathogen to uniquely adapt to thegastric mucosal environment leading to survival and long-termcolonization in humans. However, the known virulence factors are onlyassociated with increased risk of disease and are not absolute. Perhapsthe most obvious system what H. pylori developed is its powerful ureaseenzyme, which by converting urea into ammonia and carbon dioxide allowssurvival under acidic conditions. Expression of the cytoplasmicapoenzyme is constitutive and its abundance can be as high as 15% oftotal H. pylori protein. The activity of the enzyme is increased in lowpH and the conductance of the inner membrane is also increased for ureaunder low pH conditions. The produced NH₃ diffuses to the periplasmprotecting thereby the bacterium against the extremely acidicenvironment {Prinz, C. et al., 2003}. The vacuolating cytotoxin productof the vacA gene of H. pylori induces vacuole formation and, thus,perturbation of structure and function in epithelial cells. The vacAgene is present in all strains but its expression varies. It is nowknown that VacA acts through a Z-type protein tyrosine phosphatasereceptor by increasing its tyrosine phosphorylation activity on theG-protein coupled receptor kinase-interactor 1, leading to markeddetachment of gastric epithelial cells from their base membrane, apossible mechanism behind H. pylori-induced epithelial cell demise andconsequent peptic ulcer formation {Fujikawa, A. et al., 2003}. A majorvirulence-associated genetic element in H. pylori is the 40 Kbppathogenicity island, Cag (cytotoxin associated gene) PAI, contained inthe majority of strains. The PAI harbors about 30 genes and one geneproduct, CagA was originally identified in serological studies as animportant determinant of disease outcome in H. pylori infectedindividuals. The Cag PAI contains genes that have close sequencesimilarities to a type IV secretion system, known to provide a mechanismfor direct transfer of bacterial effector proteins into eukaryotic hostcells. Not surprising therefore that the CagA protein has beendemonstrated as the effector protein that translocates from adherent H.pylori into epithelial cells in vitro. The subsequently phosphorylatedCagA rearranges the host cytoskeleton, which then leads to pedestalformation adjacent to the bacteria {Bjorkholm, B. et al., 2003}.Although, it is present in strains expressing VacA, the cagA gene is notlinked chromosomally to vacA. Strains with the cagA PAI and the vacAgenotype (type I strains) are associated with higher frequency withpatients suffering from duodenal ulcer, atrophic gastritis and gastriccarcinoma compared with those lacking CagA and VacA (type R strains){Censini, S. et al., 1996}. Human populations in distinct geographicalregions can be differentiated based on genotypic variations located tothe right end of the cag PAI {Kersulyte, D. et al., 2000}.

Attachment of H. pylori to gastric epithelium is promoted by a number offactors. The Hpa hemagglutinin binds to sialic acid components oferythrocytes while the babA2-encoded BabA adhesion binds to thehisto-blood group antigen Lewis^(b), present on gastric epithelialcells. babA2-positive strains are more frequently isolated from patientswith peptic ulcer disease and gastric carcinoma than type 1 strains and,when compared to type 1 strains lacking babA2, type 1 isolates that alsoharbor the babA2 gene are more prevalent in patients with atrophicgastritis and intestinal metaplasia. The SabA adhesion protein is shownto associate with a glycoconjugate on the onco-fetal surface antigensialyl-Le^(x) that is expressed on immature cells of the developingfetal gastric epithelium as well as on rapidly proliferatingundifferentiated cells of cancerous and precancerous lesions {Dubreuil,J. et al., 2002}.

Another potent virulence-associated mechanism evolved in H. pylori isits natural competence for transformation together with the pathogen'shighest rate of recombination of any known bacterial species. Thismechanism makes H. pylori capable of acquiring new genetic material viahorizontal gene transfer, a common phenomenon during colonization of anindividual and this can result in the generation of novel pathogensubtypes (quasispecies) that exhibit profound changes in virulencemarkers, such as the cag PAI {Loughlin, M et al., 2003}. Such extremegenetic variability, with any given isolate easily distinguishable frommost others by DNA fingerprinting, has also been proposed to account forthe expression diversity of many cell surface associated or secretedproteins {Ferrero, R. et al., 2001}. Although, it is not yet clear whyonly a relatively small portion of the infected population developclinically manifest disease, the above mentioned pathogen-relatedfactors together with emerging host-specific characteristics, such asIL-1β promoter allele polymorphism, are likely contribute to the complexmechanisms that lie behind H. pylori pathogenicity {Blaser, M., 2000}.

Today, patients diagnosed with H. pylori infection are treated with acombination of one or two antibiotics and a proton pump inhibitor orbismuth. There are a number of standard combinations but re-infection(most likely from parts of the stomach where eradication did not happen)can occur. Current combinational treatment regimes reach 80-90%eradication rates in most cases but since H. pylori strains are emergingwith resistance to one or more of the antibiotics that currentlycomprise any of the treatment combinations, development of newstrategies is urgently needed for an effective treatment to prevent orameliorate H. pylori infections. A vaccine could not only preventinfections by Helicobacter, but more specifically prevent or amelioratecolonization of host tissues, thereby reducing the incidence of gastricatrophy, peptic ulcer disease and gastric cancer. Elimination of severechronic conditions would be a direct consequence of reducing theincidence of acute infection and carriage of the organism.

A vaccine can contain a whole variety of different antigens. Examples ofantigens are whole-killed or attenuated organisms, subfractions of theseorganisms/tissues, proteins, or, in their most simple form, peptides.Antigens can also be recognized by the immune system in form ofglycosylated proteins or peptides and may also be or containpolysaccharides or lipids. Short peptides can be used since for examplecytotoxic T-cells (CTL) recognize antigens in form of short usually 8-11amino acids long peptides in conjunction with major histocompatibilitycomplex (MHC). B-cells can recognize linear epitopes as short as 45amino acids, as well as three-dimensional structures (conformationalepitopes). In order to obtain sustained, antigen-specific immuneresponses, adjuvants need to trigger immune cascades that involve allcells of the immune system necessary. Primarily, adjuvants are acting,but are not restricted in their mode of action, on so-called antigenpresenting cells (APCs). These cells usually first encounter theantigen(s) followed by presentation of processed or unmodified antigento immune effector cells. Intermediate cell types may also be involved.Only effector cells with the appropriate specificity are activated in aproductive immune response. The adjuvant may also locally retainantigens and co-injected other factors. In addition the adjuvant may actas a chemoattractant for other immune cells or may act locally and/orsystemically as a stimulating agent for the immune system.

Attempts to develop a Helicobacter vaccine have focused mainly onwhole-cell and attenuated or subunit vaccine approaches. The initial“proof of principle” studies to generate an H. pylori vaccine wereperformed using inactivated whole-cell preparations and cholera toxin asa mucosal adjuvant. Although, such vaccines were highly effective ininducing protective immunity against gastric infection in mice, theirsafety and licensing as well as difficulties in producing H. pyloripreparations in vitro in large scale eliminated them from human trials{Ferrero, R. et al., 2001}; {Sutton, P., 2001}. For second generationsubunit vaccines, candidate antigens were identified by empiricalapproaches. The selection criteria for these antigens were linked toknown or suspected roles of the proteins in bacterial virulence. Suchcandidates include the urease holenzyme and its subunits, UreA and UreB,heat shock protein homologues of the chaperonins GroEL and GroES, theVacA cytotoxin and catalase (KatA) ({Prinz, C. et al., 2003};{Svennerholm, A., 2003} and references therein). Another set ofcandidate proteins were identified in subsequent studies on the basis oftheir immunoreactivity in in vitro assays. H. pylori genomic expressionlibraries were screened with antibodies from mice that had beenimmunized with H. pylori whole cell sonicates or outer membrane vesiclesin the presence of cholera toxin. Antigenic proteins were purified fromselected E. coli clones and their identity determined by N-terminalsequencing. Among known antigens, such as UreA, UreB, the GroELhomologue and Lpp20 lipoprotein, four previously uncharacterizedproteins were also identified. One had homology to L7/L12 ribosomalproteins and the rest were of unknown function. A similar screeningstrategy combined with a chimeric fusion technique confirmed the Lpp20protein as a vaccine candidate antigen {Oliaro, J. et al., 2000}. Thereare other proteins under consideration for vaccine development that arebased on recent identifications employing multiparameter selectioncriteria. These include an Hpa homologue (HP0410) and a novel protein ofunknown function (HP0231), both with high protective efficacy {Sabarth,N. et al., 2002}. Despite the benefits of both prophylactic andtherapeutic vaccination in animals as demonstrated in several studies,bacterial eradication (sterilizing immunity) has not been described inhumans.

Since the above mentioned identification methods are either empirical orlimited to a specific selection criterion, there is a demand to identifyadditional relevant antigens of H. pylori using an efficient andcomprehensive identification and validation technology.

The present inventors have developed a method for identification,isolation and production of hyperimmune serum reactive antigens from aspecific pathogen, especially from Staphylococcus aureus andStaphylococcus epidermidis (WO 02/059148). However, given thedifferences in biological property, pathogenic potency and geneticbackground, Helicobacter pylori is distinctive from Staphylococcusstrains. Importantly, the selection of sera for the identification ofantigens from H. pylori is different from that applied to the S. aureusscreens.

Three major types of human sera were collected for this purpose. First,healthy adults below <45 years of age were tested for H. pylori-specificIgG and IgA serum antibody levels by ELISA using total bacterial lysateand culture supernatant proteins. High titer individuals wereinterviewed and seleted based on the absence of medical history,symptoms or complains related to H. pylori diseases. Based oncorrelative data, protective (colonization neutralizing) antibodies arelikely to be present in exposed individuals who are not carriers of H.pylori or not susceptible to disease casued by H. pylori. High titersera from symptom-free healthy adults were included in the genomic basedantigen identification. This approach for selection of human sera isbasically very different from that used for S. aureus, where carriage ornoncarriage state cannot be associated with antibody levels.

Second, serum samples from patients with gastric cancer werecharacterized for anti-H. pylori antibody titers using ELISA and hightiter sera were selected for the screens. The third group of serumsamples was obtained from individuals with duodenal ulcer and high titersera determined by ELISA were selected for the screens.

The genomes of the two bacterial species H. pylori and S. aureus byitself show a number of important differences. The genome of H. pyloricontains approximately 1.65 Mb sequence information, while S. aureusharbours about 2.85 Mb. They have an average GC content of 39 and 33%,respectively. In addition, the two bacterial species require differentgrowth conditions and media for propagation. While H. pylori is astrictly human pathogen, S. aureus can also be found infecting a rangeof warm-blooded animals. A list of the most important diseases, whichcan be inflicted by the two pathogens, is presented below. S. aureuscauses mainly nosocomial, opportunistic infections: impetigo,folliculitis, abscesses, boils, infected lacerations, endocarditis,meningitis, septic arthritis, pneumonia, osteomyelitis, scalded skinsyndrome (SSS), toxic shock syndrome. H. pylori causes likely communityaquired gastro-intestinal infections: self limiting traveler's diarrhea,corpus predominant or pangastritis, peptic ulcer disease (stomach andduodenum), gastric cancer (adenocarcinoma), chronic atrophic gastritis(CAG) and MALT (mucosa-associated lymphoid tissue, non-Hodgkin's type Bcell lymphoma).

The problem underlying the present invention was to provide means forthe development of medicaments such as vaccines against H. pyloriinfection. Particularly, the problem was to provide an efficient,relevant and comprehensive set of nucleic acid molecules or antigensfrom H. pylori that can be used for the manufacture of said medicaments.

Therefore, the present invention provides an isolated nucleic acidmolecule encoding a hyperimmune serum reactive antigen or a fragmentthereof comprising a nucleic acid sequence, which is selected from thegroup consisting of:

-   -   a) a nucleic acid molecule having at least 70% sequence identity        to a nucleic acid molecule selected from Seq ID No 3-4, 16,        19-21, 28-29, 33-38, 41-42, 44, 48-52, 55, 57-58, 61, 63, 65,        67-68, 72, 74-75, 81, 84, 91, 94, 96-97, 101, 105-108, 112,        115-117, 119, 123-178.    -   b) a nucleic acid molecule which is complementary to the nucleic        acid molecule of a),    -   c) a nucleic acid molecule comprising at least 15 sequential        bases of the nucleic acid molecule of a) or b)    -   d) a nucleic acid molecule which anneals under stringent        hybridisation conditions to the nucleic acid molecule of a), b),        or c)    -   e) a nucleic acid molecule which, but for the degeneracy of the        genetic code, would hybridise to the nucleic acid molecule        defined in a), b), c) or d).

According to a preferred embodiment of the present invention thesequence identity is at least 80%, preferably at least 95%, especially100%.

Furthermore, the present invention provides an isolated nucleic acidmolecule encoding a hyperimmune serum reactive antigen or a fragmentthereof comprising a nucleic acid sequence selected from the groupconsisting of

-   -   a) a nucleic acid molecule having at least 96% sequence identity        to a nucleic acid molecule selected from Seq ID No 8-10, 13-15,        17-18, 24, 27, 32, 39-40, 45-47, 56, 59, 62, 69-70, 73, 77, 79,        82, 85-86, 88, 90, 103, 109-110, 114, 121,    -   b) a nucleic acid molecule which is complementary to the nucleic        acid molecule of a),    -   c) a nucleic acid molecule comprising at least 15 sequential        bases of the nucleic acid molecule of a) or b)    -   d) a nucleic acid molecule which anneals under stringent        hybridisation conditions to the nucleic acid molecule of a), b)        or c),    -   e) a nucleic acid molecule which, but for the degeneracy of the        genetic code, would hybridise to the nucleic acid defined in a),        b), c) or d).

According to another aspect, the present invention provides an isolatednucleic acid molecule comprising a nucleic acid sequence selected fromthe group consisting of

-   -   a) a nucleic acid molecule selected from Seq ID No 5, 7, 30-31,        53, 60, 66, 76, 83, 87, 92, 99, 120,    -   b) a nucleic acid molecule which is complementary to the nucleic        acid of a),    -   c) a nucleic acid molecule which, but for the degeneracy of the        genetic code, would hybridise to the nucleic acid defined in a),        b), c) or d).

Preferably, the nucleic acid molecule is DNA or RNA.

According to a preferred embodiment of the present invention, thenucleic acid molecule is isolated from a genomic DNA, especially from aH. pylori genomic DNA.

According to the present invention a vector comprising a nucleic acidmolecule according to any of the aspects of the present invention isprovided.

In a preferred embodiment the vector is adapted for recombinantexpression of the hyperimmune serum reactive antigens or fragmentsthereof encoded by the nucleic acid molecule according to the presentinvention.

The present invention also provides a host cell comprising the vectoraccording to the present invention.

According to another aspect the present invention further provides ahyperimmune serum-reactive antigen comprising an amino acid sequencebeing encoded by a nucleic acid molecule according to the presentinvention.

In a preferred embodiment the amino acid sequence (polypeptide) isselected from the group consisting of Seq ID No 181-182, 194, 197-199,206-207, 211-216, 219-220, 222, 226-230, 233, 235-236, 239, 241, 243,245-246, 250, 252-253, 259, 262, 269, 272, 274-275, 279, 283-286, 290,293-295, 297, 301-356.

In another preferred embodiment the amino acid sequence (polypeptide) isselected from the group consisting of Seq ID No 186-188, 191-193,195-196, 202, 205, 210, 217-218, 223-225, 234, 237, 240, 247-248, 251,255, 257, 260, 263-264, 266, 268, 281, 287-288, 292, 299.

In a further preferred embodiment the amino acid sequence (polypeptide)is selected from the group consisting of Seq ID No 183, 185, 208-209,231, 238, 244, 254, 261, 265, 270, 277, 298.

According to a further aspect the present invention provides fragmentsof hyperimmune serum-reactive antigens selected from the groupconsisting of peptides comprising amino acid sequences of column“predicted immunogenic aa” and “location of identified immunogenicregion” of Table 1, the serum reactive epitope of Table 3 especiallypeptides comprising amino acids 63-91, 95-101, 110-116, 134-148,150-156, 158-164, 188-193, 197-209, 226-241, 247-254, 291-297, 312-319,338-346, 351-358, 366-378, 404-410, 420-438, 448-454, 465-473, 482-488,490-498, 503-510, 512-519, 531-543, 547-554, 568-575, 589-604, 610-631and 239-308 of Seq ID No 179; 16-29, 35-47, 50-68, 70-79, 91-101,143-149, 158-163, 185-191, 196-206, 215-224, 230-237, 244-251, 258-278,290-311, 319-325, 338-351, 365-385, 396-429, 445-454, 458-466, 491-499,501-521, 17-79 and 218-233 of Seq ID No 180; 4-10, 16-41, 46-66, 77-84,91-97, 102-118, 125-144, 187-200, 202-214, 245-253, 255-261, 286-295,300-330, 335-342, 350-361, 363-381, 385-392, 396-416, 435-450 and460-470 of Seq ID No 181; 11-19, 27-48, 52-59, 77-82, 84-107, 118-125,127-154, 178-183, 192-209, 215-221, 286-295, 302-313, 350-357, 402-415,417-431, 453-463, 465-493 and 313-331 of Seq ID No 182; 19-26, 30-43,47-55, 63-68, 72-80, 97-104, 107-119, 129-146, 160-175, 194-216,231-251, 254-260 and 26-43 of Seq ID No 183; 7-13, 29-37, 65-81,110-120, 123-131, 135-152, 230-249, 254-260, 284-290, 292-299, 317-326,329-336, 403-444, 452-458, 466-477, 490-498, 510-519, 541-550, 557-566and 533-567 of Seq ID No 184; 5-47, 71-77, 79-86, 89-95, 120-126,137-144, 176-181, 184-196, 202-208, 211-232, 236-282, 301-313, 317-325,341-347, 353-384, 394-400, 412-433, 436-443 and 59-75 of Seq ID No 185;4-18, 22-38, 59-69, 106-112, 116-130, 138-149, 156-170, 175-197,200-214, 216-223, 233-244, 255-261, 266-276, 279-286, 325-333, 342-348,366-399, 402-420, 429-441, 1-104 and 130-147 of Seq ID No 186; 50-58,69-95, 97-113, 131-136, 157-163, 170-175, 188-212, 220-226, 254-259,265-277, 283-289, 297-308, 311-318, 347-358, 360-369, 378-401, 416-421,440-450, 454-462, 470-476, 493-502, 506-514, 536-567, 585-590, 598-607,613-618, 653-659 and 35-46 of Seq ID No 187; 16-29, 32-60, 65-87,89-123, 128-134, 137-158, 162-173, 178-196, 210-216, 218-228 and 206-225of Seq ID No 188; 10-20, 26-35, 51-64, 86-91, 94-100, 113-122, 154-160,185-191, 193-201, 211-217, 225-230, 237-246, 251-257, 298-304, 306-312,316-328, 340-348, 357-389, 391-397, 415-421, 449-456, 458-471, 488-495,502-511, 24-55 and 236-341 of Seq ID No 189; 5-22, 41-51, 87-93,114-122, 127-136, 150-156, 158-166, 223-233, 245-263, 291-296, 9-126 and127-285 of Seq ID No 190; 30-43, 46-56, 61-70, 72-83, 85-93, 103-113,119-125, 151-166, 179-191, 212-218, 225-231, 236-243, 262-267, 291-307,331-344, 349-355, 366-372, 380-386, 414-422, 428-447, 459-464, 469-478,507-519, 525-544, 563-569, 576-590, 620-626, 633-643, 654-659, 665-671,684-707, 717-723, 725-733, 747-779, 782-801 and 347-361 of Seq ID No191; 4-12, 14-26, 37-80, 107-115, 133-139, 144-150, 154-165, 173-180,191-199, 205-211, 221-231, 237-244, 254-284, 307-340, 342-353, 360-368,370-380, 479-493, 495-503, 509-522, 525-536, 539-547, 554-560, 565-573,578-583, 7-23 and 465-479 of Seq ID No 192; 4-17, 47-55, 76-83, 85-100,104-112, 117-123, 126-135, 142-148, 156-167, 174-182, 267-273 and258-283 of Seq ID No 193; 8-32, 36-42, 65-88, 102-108, 112-140, 147-163,170-179, 183-193 and 117-124 of Seq ID No 194; 12-18, 45-50, 62-77,82-95, 99-113, 115-123, 125-147, 155-177, 187-209, 211-223, 244-253,259-270, 278-297, 302-307, 311-318, 329-334, 350-356, 359-365, 390-400,402-413 and 333-350 of Seq ID No 195; 4-13, 15-27, 30-46, 53-58, 68-74,82-95, 115-126, 134-139, 148-153, 159-176, 182-199, 201-217, 220-225,227-235, 237-248, 253-266, 300-315, 322-336, 390-396, 412-426, 438-445,448-459, 477-484, 502-508, 515-527, 529-537, 553-568, 643-651, 658-667,690-703 and 376-400 of Seq ID No 196; 4-10, 24-32, 38-55, 59-67, 70-77,80-87, 89-97, 123-129, 134-151, 166-172, 178-189, 191-216, 218-235,245-259, 271-315, 326-339, 341-360 and 73-94 of Seq ID No 197; 13-25,31-3, 43-57, 79-85, 92-99, 106-112, 117-128, 130-139, 146-158, 160-175,194-204, 211-222, 225-232, 234-242, 263-270, 278-292, 299-320, 322-333and 240-256 of Seq ID No 198; 4-17, 55-63, 66-101, 109-131, 135-143,145-151, 155-161, 164-170, 177-185, 192-198, 213-218, 223-238, 246-256,258-268, 273-283, 309-314, 322-328 and 195-221 of Seq ID No 199; 13-24,31-39, 41-50, 63-69, 90-96, 104-109, 116-141, 148-153, 161-167, 173-178,190-209, 253-258, 265-272, 279-289, 295-312, 317-343, 355-366, 376-389,400-407, 430-451, 453-464, 466-472, 487-493, 499-505, 523-538, 554-559,568-579, 584-01 and 344-363 of Seq ID No 200; 5-22, 30-36, 53-59, 61-70,82-92, 99-106, 120-131, 135-148, 154-167, 169-183, 187-199, 204-212,231-247 and 111-249 of Seq ID No 201; 17-36, 40-66, 71-144, 148-171,173-191, 199-214, 220-252, 265-272, 278-288, 298-333, 342-385 and287-307 of Seq ID No 202; 4-16, 22-28, 30-36, 42-48, 95-116, 154-162,164-174, 239-252, 258-263, 273-285, 306-313, 323-333, 341-357, 363-369,372-379, 395-401, 430-436, 438-453, 464-480, 33-44, 233-258 and 349-369of Seq ID No 203; 4-21, 30-37, 46-53, 59-68, 80-92, 98-104, 118-143,150-160, 165-185, 187-200, 204-211, 224-236, 241-246, 252-258, 271-280,288-294, 311-320, 335-341 and 191-350 of Seq ID No 204; 4-16, 37-59,64-70, 79-87, 93-102, 107-127, 143-165, 172-188, 197-204, 207-218,221-227, 242-248, 258-277, 289-296, 298-316, 332-338, 344-365, 367-373,375-382, 400-408, 415-425, 438-446 and 235-250 of Seq ID No 205; 4-37,39-66, 84-98, 101-127, 140-149, 157-163, 166-172, 175-182, 184-193,203-208, 215-232, 234-247, 250-299, 303-345 and 183-204 of Seq ID No206; 10-20, 41-61, 73-87, 112-141, 176-192, 194-201, 205-222, 230-237,257-264, 276-282, 284-310, 312-318, 330-337, 349-357 and 304-328 of SeqID No 207; 4-31, 42-103, 105-113, 121-153, 160-181, 188-196, 210-226,231-264, 272-287, 297-304, 328-336 and 304-318 of Seq ID No 208; 21-43,46-52, 54-70, 72-79, 94-107, 133-141, 160-166, 217-253, 311-317,359-365, 374-381, 390-395, 434-440, 488-494, 497-502, 511-522, 554-563,565-574, 577-585, 591-598, 601-606, 617-625, 633-643, 658-664, 676-682,694-702, 710-719, 754-760, 782-788, 802-808, 916-921, 942-948, 955-964,973-979, 992-998, 1006-1011, 1016-1023, 1030-1038, 1046-1053, 1059-1066,1088-1098, 1119-1126, 1129-1135, 1156-1171, 1173-1181, 1202-1210,1255-1261, 1268-1280, 1295-1310, 1312-1320, 1375-1381, 1406-1417,1450-1471, 1478-1492, 1498-1506, 1569-1578, 1603-1608, 1611-1624,1648-1655, 1663-1670, 1680-1698, 1702-1707, 1713-1719, 1737-1742,1747-1753, 1762-1769, 1771-1785, 1790-1804, 1811-1818, 1830-1836,1838-1852, 1874-1886, 1893-1899, 1902-1909, 1942-1948, 1952-1962,1980-1986, 2001-2017, 2020-2028, 2042-2050, 2052-2068, 2074-2079,2083-2095, 2107-2113, 2147-2155, 2177-2194, 2203-2211, 2236-2241,2251-2258, 2267-2274, 2285-2292, 2314-2328, 2330-2340, 2358-2365,2390-2401, 2408-2418, 2432-2453, 2463-2476, 2486-2507, 2528-2537,2540-2548, 2552-2558, 2568-2576, 2596-2601, 2610-2622, 2629-2638,2653-2669, 2718-2727, 2749-2767, 2777-2784, 2789-2795, 2806-2815,2817-2824, 2835-2843, 2847-2854, 2860-2881, 511-523, 612-630 and1790-1803 of Seq ID No 209; 4-54, 61-68, 72-82, 86-93, 100-108, 115-130,147-154, 187-194, 196-207, 224-229, 236-251, 275-287 and 96-109 of SeqID No 210; 31-39, 62-69, 91-101, 158-172, 175-180, 186-193, 201-208,210-223, 243-250, 273-286, 293-299, 319-325, 343-354, 356-365, 368-384,414-435, 471-491, 512-518, 550-556, 567-581, 584-589, 633-639, 680-692,697-708, 716-721, 747-754, 779-786, 810-816 and 366-503 of Seq ID No211; 5-20, 22-48, 57-65, 96-101, 111-122, 130-145, 154-164, 170-181,193-199, 201-216, 224-241, 244-262, 281-323, 342-351, 359-367, 369-396,406-416, 424-433, 450-456, 485-491, 493-499, 501-515, 517-535 and289-305 of Seq ID No 212; 4-17, 22-44, 53-60, 66-83, 87-94, 101-106,110-116, 131-137, 148-183, 189-207, 209-215, 233-242, 251-262, 264-272,290-296, 308-327, 359-373, 375-380, 397-405, 415-420, 426-433, 444-475,478-484, 529-536, 548-558 and 106-126 of Seq ID No 213; 4-38, 42-50,58-64, 72-81, 92-118, 140-146, 157-165, 172-192, 198-204, 208-216,227-234, 238-258, 271-278, 288-293, 311-322, 327, 346, 357-370, 375-383,395-409, 411-417, 425-432, 436-445, 109-129 and 370-380 of Seq ID No214; 23-30, 36-49, 52-64, 86-94, 97-104, 121-129, 257-272, 279-286,288-294, 307-327, 334-340, 369-375, 377-386, 406-412, 418-423, 430-438,441-447, 459-465, 469-476, 482-488, 510-546, 550-580, 584-622, 638-645,653-659, 675-63, 692-705, 723-731, 752-761, 788-795 and 572 of Seq ID No215; 11-33, 36-46, 88-104, 116-126, 134-170, 189-195, 199-217, 225-250,255-261, 266-273, 280-291, 296-313, 334-341, 343-349, 354-360, 362-369,373-380, 387-401, 406-420 and 259-273 of Seq ID No 216; 9-14, 28-44,57-64, 72-79, 86-93, 104-111, 116-126, 142-150, 159-164 and 61-86 of SeqID No 217; 10-17, 26-33, 43-61, 69-95, 101-107, 109-125, 129-135,137-144, 147-153, 158-169, 177-187, 209-219, 221-232, 235-247, 261-268,271-282, 296-302, 306-347, 355-362, 364-379, 386-399, 409-418, 424-442,451-460, 467-479, 490-498 and 60-74 of Seq ID No 218; 8-14, 20-31,65-84, 94-99, 154-179, 193-207, 238-253 and 96-118 of Seq ID No 219;4-24, 30-44, 47-62, 84-93, 108-116, 124-133, 136-141, 201-209, 217-223,228-235, 238-245, 247-270, 275-285, 290-314, 328-338, 342-349, 353-365,375-383, 386-392, 394-402, 417-427, 443-459, 465-481, 492-514, 516-524,550-566, 602-617, 630-639, 666-676, 687-693, 719-730, 747-753, 783-790,799-816, 824-831, 837-842 and 167-189 of Seq ID No 220; 6-15, 18-28,58-66, 84-101, 106-129, 136-151, 154-165, 182-203, 205-211, 214-220,222-228, 233-240, 251-260, 270-277, 284-291, 306-315, 322-328, 363-369,378-388, 392-405, 443-452, 495-501, 512-523, 574-583 and 362-375 of SeqID No 221; 5-25, 27-34, 47-59, 64-70, 76-86, 145-158, 166-183, 189-202,217-231, 235-242, 260-270, 278-309 and 1-102 of Seq ID No 222; 4-19,24-76, 78-83, 90-99, 102-109, 114-122, 137-147, 154-174, 177-188,203-212, 217-223, 227-239 and 226-325 of Seq ID No 223; 7-37, 71-90,94-109, 117-128, 141-153, 179-192, 199-206, 225-231, 237-243, 258-264and 40-51 of Seq ID No 224; 13-19, 25-30, 46-59, 75-91, 101-107,114-124, 129-135, 137-145, 160-167, 171-179, 187-194, 209-215, 217-222,229-239, 243-249, 257-265, 269-275, 299-308, 310-327 and 282-300 of SeqID No 225; 86-100, 216-230, 342-369, 382-388, 424-430, 438-445, 452-458,488-494, 501-518, 554-560, 568-574, 584-592, 603-609, 611-629, 639-645,652-661, 669-699, 708-714, 726-738, 747-753, 763-775, 785-791, 794-807,815-824, 826-845, 854-860, 863-868, 870-883, 892-898, 901-906, 909-921,930-937, 946-959, 968-974, 977-990, 998-1007, 1009-1027, 1037-1043,1046-1051, 1053-1066, 1075-1081, 1084-1089, 1092-1103, 1113-1119,1122-1135, 1143-1152, 1154-1172, 1182-1188, 1191-1196, 1200-1210,1220-1226, 1229-1235, 1237-1249, 1259-1265, 1268-1281, 1289-1298,1305-1318, 1328-1334, 1337-1343, 1345-1357, 1367-1373, 1390-1396,1405-1411, 1418-1423, 1426-1435, 1445-1455, 1474-1483, 1493-1500,1505-1512, 1517-1524, 1538-1544, 1568-1578, 1595-1601, 1674-1682,1687-1720, 1728-1736, 1738-1744, 1754-1761, 1764-1774, 1798-1824,1836-1842, 1886-1893, 1895-1903, 366-781, 782-1518 and 1731-1747 of SeqID No 226; 4-17, 20-39, 46-55, 60-66, 102-110, 114-122, 125-131,161-167, 172-178, 185-190, 195-202, 218-232, 236-252, 264-291, 293-302,309-315, 324-339 and 169-381 of Seq ID No 227; 5-10, 13-40, 42-53,69-75, 83-89, 120-135, 150-161, 174-190, 203-225, 229-247, 257-287,318-348 and 30-200 of Seq ID No 228; 7-19, 43-53, 64-72, 124-139, 52-84and 120-131 of Seq ID No 229; 12-19, 39-48, 58-100, 117-123, 154-162,164-187, 189-195, 202-216, 218-235, 241-246, 262-278, 315-328, 333-347,354-366, 372-379, 391-405, 422-429, 431-442, 444-450, 458-466, 478-485,494-501, 504-510, 520-535, 573-580, 589-598, 615-625, 666-676, 686-698,722-729, 737-746, 756-767, 787-796, 805-816, 824-829, 833-848, 856-864,866-876, 879-886, 898-904, 918-924, 927-934, 941-960, 967-978 and561-575 of Seq ID No 230; 11-29, 49-55, 70-77, 84-100, 102-112, 148-155,160-177, 181-204 and 1-104 of Seq ID No 231; 27-44, 64-71, 122-133,151-156, 164-178, 214-220, 226-232, 235-244, 253-262, 282-288, 294-310,317-325, 350-356, 362-368, 376-383, 438-443, 449-454, 459-464, 492-498,500-511, 529-535, 538-546, 567-573, 597-603, 660-665, 674-679, 724-734,763-769, 773-784, 791-801, 807-815, 821-826, 840-848, 863-868, 897-902,908-928, 932-953, 956-975, 980-987, 990-996, 1012-1018, 1042-1063,1095-1116, 1149-1157, 1160-1167, 110-357, 358-501 and 502-1161 of Seq IDNo 232; 4-21, 64-71, 73-84, 128-138, 144-162, 203-217, 240-263, 288-298,300-308, 310-317, 325-351, 369-380, 391-411 and 330-345 of Seq ID No233; 5-11, 25-31, 39-48, 51-79, 89-98, 100-122, 135-148, 166-201,203-227, 230-250, 254-260, 266-272, 274-282, 299-305, 328-337 and 31-4of Seq ID No 234; 12-23, 29-48, 51-60, 66-72, 75-81, 83-93, 103-115,133-148, 168-174, 195-204, 222-229, 231-240, 242-251, 270-280, 286-305,322-344, 349-360, 364-370, 378-400, 421-441, 448-484, 486-493, 495-501,504-534, 547-561, 567-590, 597-607, 621-635, 643-649, 658-685, 688-694,702-711, 717-731, 737-742, 759-765, 767-772, 776-786, 803-809, 815-825,854-908, 910-919, 923-930, 942-948, 961-975, 994-1014 and 915-940 of SeqID No 235; 4-9, 32-47, 51-61, 75-96, 139-191 and 1-124 of Seq ID No 236;4-13, 17-38, 43-49, 55-76, 88-95, 110-121, 128-146, 151-157, 162-214,222-240, 243-249, 251-273, 275-281, 292-298, 300-309, 312-320, 322-331,355-369, 376-408, 446-460, 471-482, 485-509 and 191-203 of Seq ID No237; 4-21, 72-82, 89-103, 106-115, 118-124, 140-146, 174-184, 191-200,204-213, 218-224, 261-266, 282-293, 299-309, 311-340, 342-358, 362-372,381-389, 391-402, 413-421, 438-447, 457-464, 470-478, 501-507, 545-560,578-624, 631-641, 658-670, 680-689, 717-738, 753-759, 795-805, 816-822,830-838, 842-848, 869-881, 892-898, 33-51 and 818-835 of Seq ID No 238;4-21, 79-85, 156-177, 183-188, 206-214, 243-249, 261-269, 287-292,315-322, 334-345, 360-366, 374-390, 402-411, 37-97 and 260-399 of Seq IDNo 239; 4-9, 19-54, 58-78, 97-104, 111-120, 126-134, 137-145, 163-173,178-188, 193-203, 211-224, 246-286, 288-324, 337-346, 355-362, 374-390,392-398, 409-417 and 240-249 of Seq ID No 240; 5-12, 14-31, 35-41,43-61, 82-92, 97-105, 134-145, 155-166, 184-203, 215-223, 225-251,272-279, 281-306, 310-345, 358-418, 435-473, 482-490, 525-532, 538-547,549-563, 578-604, 613-639 and 144-154 of Seq ID No 241; 53-59, 64-72,74-100, 133-152, 154-172, 176-181, 207-214, 225-238, 275-297, 304-310,331-340, 362-367, 384-395, 403-410, 437-443, 448-456, 482-490, 579-597,602-610, 625-630, 633-651, 699-707, 709-715, 734-743, 750-762 and544-685 of Seq ID No 242; 12-18, 22-40, 45-83, 89-97, 103-109, 147-153,159-173, 195-204, 210-219, 243-253, 259-265, 273-282, 303-309, 315-325,332-340, 346-358, 362-37, 377-390, 393-402, 418-426, 447-455, 467-480,505-512, 514-525, 548-561, 566-576, 584-596, 619-626, 638-645, 649-659,661-680, 699-708, 714-720, 753-759, 766-772, 775-781, 801-808, 202-218,282-299, 339-350 and 617-628 of Seq ID No 243; 5-33, 52-62, 87-101,111-135, 137-143, 145-152, 190-202, 209-221, 233-245, 253-270 and151-215 of Seq ID No 244; 19-29, 32-39, 42-48, 75-94, 124-135, 137-145,152-160, 176-182, 193-203, 215-236, 266-273, 275-291, 297-306, 311-319,322-342, 348-360, 369-378, 394-401 and 48-64 of Seq ID No 245; 4-11,13-33, 36-43, 53-63, 65-80, 112-129, 134-141, 143-155, 157-168, 178-188,191-199, 201-207, 215-229, 242-255, 263-270, 283-315, 320-329, 333-338,340-349, 412-426, 465-478, 455-490, 498-512, 540-554 and 390-516 of SeqID No 246; 4-18, 23-32, 41-47, 54-70, 88-99, 104-111, 118-138, 143-148,150-162, 168-175, 181-188, 203-211, 214-220, 227-245, 251-268, 275-281,287-296, 323-333 and 1-90 of Seq ID No 247; 8-34, 38-49, 72-83, 85-91,94-104, 112-125, 134-142, 148-168, 181-189, 191-198, 202-214, 222-233,242-254, 256-262, 273-278, 287-294, 314-325 and 141-159 of Seq ID No248; 4-24, 30-36, 47-75, 82-105, 124-134, 151-157, 192-202, 208-214,219-226, 234-247, 285-290, 318-324, 332-340, 343-349, 380-386, 453-462,472-478, 484-501, 531-540, 550-557, 604-612, 620-625, 642-648, 652-671,64-84, 93-180 and 181-446 of Seq ID No 249; 12-18, 24-32, 68-75, 77-83,96-101, 109-116, 129-136, 152-164, 175-184, 190-199, 206-215, 224-233,241-250, 258-264, 273-292, 302-312, 319-331, 334-346, 348-368, 387-395,408-416, 420-429, 437-452 and 364-374 of Seq ID No 250; 11-28, 36-52,60-67, 74-79, 108-116 and 61-76 of Seq ID No 251; 20-27, 38-49, 69-74,84-107, 138-145, 161-168, 179-195, 210-226, 228-252, 267-281, 283-296,305-311, 333-340, 342-356, 361-372, 380-399, 401-414, 458-466, 475-481,492-507, 515-520 and 146-160 of Seq ID No 252; 43-61, 68-74, 76-90,120-128, 130-149, 156-161, 164-182, 206-234, 242-252, 269-274, 291-304,332-345, 349-355, 360-371, 374-388, 434-440, 447-453, 459-465, 469-496,504-522 and 261-285 of Seq ID No 253; 4-17, 24-30, 37-49, 87-98,118-124, 126-136, 144-171, 176-188, 206-214, 216-228, 233-240, 246-252,262-271, 277-297, 307-330, 333-342, 346-352, 355-361, 368-386, 391-400,413-420, 474-480 and 401-427 of Seq ID No 254; 15-26, 31-46, 51-72,80-93, 96-109, 131-137, 150-158, 179-185, 189-209, 211-219, 221-234,241-247, 255-262, 265-271, 283-288 and 173-190 of Seq ID No 255; 28-37,39-45, 51-58, 77-84, 89-97, 132-148, 171-180, 199-205, 212-218, 220-226,257-265, 273-300, 307-327, 334-340, 344-365, 385-390, 402-408, 426-436,450-468, 476-485 and 425-497 of Seq ID No 256; 4-25, 70-76, 80-88,90-100, 120-128, 162-169, 183-203, 261-277, 279-289, 291-297, 302-308,321-327, 339-353, 358-377, 392-401, 404-410, 414-422, 443-450, 456-461,470-488, 490-497, 510-535, 570-611, 618-630, 639-647, 649-660, 668-690,702-716, 718-724, 737-747, 750-764 and 497-509 of Seq ID No 257; 12-48,50-64, 99-108, 216-223, 235-241, 244-254, 262-274, 287-293, 310-316,320-326, 361-366, 377-383, 390-395, 408-414, 418-425, 438-444, 462-469,494-505, 524-530, 536-547, 551-566, 592-598, 601-613, 678-685, 687-695,709-717, 727-737, 751-757, 760-765, 772-778, 782-788, 801-807, 822-830,859-868, 870-878, 884-890, 898-903, 909-919, 953-969, 973-980, 990-1000,1002-1019, 1041-1047, 1059-1065, 1090-1095, 1116-1127, 1130-1139,1143-1149, 1151-1168, 1178-1183, 1188-1195, 1197-1209, 1213-1220,1226-1234, 1236-1247, 1255-1274, 1276-1282, 76-100, 270-284, 309-438,493-505, 786-942 and 947-967 of Seq ID No 258; 4-9, 24-34, 46-95,97-109, 119-130 and 138-156 of Seq ID No 259; 9-26, 28-35, 43-53, 55-68,83-92, 99-105, 110-135, 139-149, 157-162, 164-170, 173-183, 193-208,210-230, 239-245, 253-259, 263-271, 293-305, 310-320, 322-331, 336-343,351-364, 367-376, 92-107 and 154-173 of Seq ID No 260; 19-39, 52-62,108-117, 145-152, 160-168, 194-203, 229-240, 252-268, 280-287, 308-316,333-339, 383-390, 403-412, 414-424, 438-445, 464-472, 479-484, 489-505,510-526 and 247-260 of Seq ID No 261; 5-17, 25-52, 60-77, 105-113,118-125, 162-167, 228-234, 272-279, 328-334, 341-357, 381-395, 400-406,512-518, 557-569, 586-592, 645-651, 690-695, 701-709, 720-726, 733-743,751-758, 781-786, 879-886, 929-934, 939-944, 952-960, 965-975, 994-1001,1039-1045, 1102-1109, 1164-1181, 1198-1206, 1223-1229, 1253-1259,1283-1292, 1312-1317, 1339-1349, 1360-1370, 1389-1398, 1400-1412,1452-1465, 1470-1484, 1490-1497, 1519-1525, 1554-1564, 1578-1591,1623-1636, 1638-1646, 1669-1679, 1685-1697, 1704-1711, 1713-1720,1730-1736, 1738-1749, 1756-1764, 1778-1786, 1796-1803, 1817-1826,1849-1866, 1975-1993, 2017-2032, 2044-2053, 2070-2086, 2091-2109,2116-2127, 2156-2167, 2182-2188, 2197-2202, 2244-2252, 2281-2287,2290-2307, 2350-2361, 2383-2404, 2425-2433, 2445-2455, 2495-2505 and394-549 of Seq ID No 262; 9-24, 31-53, 57-67, 69-79, 84-114, 133-141,144-172, 178-186 and 13-46 of Seq ID No 263; 4-25, 27-35, 43-52, 59-70,79-91, 115-130, 136-152, 154-163, 170-179 and 1-58 of Seq ID No 264;4-30, 49-55, 71-80, 96-105, 111-126, 139-146, 149-162, 239-245, 279-285,290-296, 300-307, 331-337, 343-350 and 250-351 of Seq ID No 265; 9-27,34-41, 43-51, 92-111, 114-120, 123-131, 139-150, 156-171, 176-186,188-204, 229-241, 252-258, 266-279, 288-297, 319-334, 338-348, 373-379,389-398, 431-439, 479-484 and 214-398 of Seq ID No 266; 4-15, 18-27,47-52, 68-83, 91-97, 104-110, 115-121, 139-147, 157-164, 198-206,227-236, 241-254, 264-273, 278-289, 311-320, 353-361, 372-383, 405-420,426-434 and 232-386 of Seq ID No 267; 4-10, 24-34, 91-97, 129-141,156-163, 184-190, 205-219, 229-235, 256-273, 278-285 and 93-116 of SeqID No 268; 7-29, 35-54, 71-83, 85-91, 104-111, 122-134, 138-144,146-154, 158-174, 177-183, 186-201, 207-215, 223-235, 240-247, 262-273,275-283, 287-292 and 48-66 of Seq ID No 269; 7-27, 31-47, 49-70, 75-102,110-149, 157-171, 217-223, 235-251, 294-302, 358-364, 367-375, 387-393,395-412, 423-430, 441-451, 456-470, 472-486, 488-495, 499-509, 515-529,536-549, 556-570, 574-603, 607-615, 625-633, 642-658, 670-676, 683-702,708-716, 720-726, 747-756, 763-784, 803-812, 815-826 and 475-490 of SeqID No 270; 7-22, 30-38, 53-59, 64-75, 83-95, 97-112, 120-131, 133-142,145-151, 154-166, 172-180, 189-203, 227-238, 277-287, 9-156 and 174-287of Seq ID No 271; 13-23, 25-32, 111-117, 150-164, 185-193, 207-212,216-224, 230-236, 263-272, 304-311, 342-348, 374-385, 391-407, 444-458,480-487, 489-499, 523-542, 544-558, 572-579, 620-640, 686-696, 703-710,742-755, 765-772, 817-822, 830-837, 865-872, 931-937 and 66-86 of Seq IDNo 272; 4-27, 49-56, 62-70, 86-92, 121-127, 151-163, 170-182, 195-202,212-226, 237-243 and 234-254 of Seq ID No 273; 4-10, 13-24, 39-51,62-78, 92-104, 107-117, 134-141, 156-161, 166-181, 210-216, 222-229,256-266, 273-280, 297-304, 313-330, 336-349, 371-376, 433-439, 443-448,488-493, 506-515, 527-534, 560-572, 575-583, 587-593 and 252-483 of SeqID No 274; 4-15, 21-38, 45-56, 81-95, 102-108, 118-130, 133-147,152-162, 166-171, 199-204, 211-218, 230-240, 253-261, 274-283, 288-294,312-317, 325-336, 344-357, 391-414 and 24-146 of Seq ID No 275; 26-31,38-56, 65-82, 90-101, 112-119, 123-153, 175-188, 197-216, 234-242,249-265, 273-286, 290-305, 327-335, 338-346, 361-372, 394-404 and290-306 of Seq ID No 276; 17-26, 43-48, 50-73, 81-93, 95-107, 139-146,158-168, 171-176, 190-196, 202-212, 216-223, 243-266, 274-282, 308-313,324-330, 344-378, 380-387, 403-422, 427-443, 448-455, 457-465, 491-515,517-528, 553-567, 589-599, 610-617, 642-648, 670-697, 709-717, 726-743,745-759, 769-803, 807-823, 840-849 and 820-851 of Seq ID No 277; 4-18,39-48, 53-63, 66-90, 102-117, 125-134, 137-145, 156-162, 169-197, 26-40and 56-80 of Seq ID No 278; 21-33, 36-42, 49-60, 68-76, 91-105, 123-130,141-161, 169-178, 185-190, 192-199, 205-214, 223-233, 239-247, 260-269,284-293, 300-314, 324-352, 357-364, 373-382, 389-403, 420-432, 438-446,466-471, 477-484, 503-509, 549-556, 558-576, 600-623, 625-635, 654-661,663-669, 671-687, 702-716, 735-741, 744-750, 757-766, 776-786, 807-815,824-832, 854-860, 863-897, 909-915, 920-946, 952-959, 982-997,1024-1038, 1049-1055, 1071-1085, 1104-1113, 1121-1132, 1138-1150,1187-1196, 1212-1221, 1227-1236, 1257-1262, 1264-1278, 1282-1294,1307-1318, 1353-1370, 1382-1388, 1396-1409, 1434-1440, 1446-1454,1465-1478, 1485-1513, 1516-1529, 1540-1545, 1563-1568, 1575-1593,1607-1616, 1628-1645, 1648-1661, 1676-1682, 1689-1697, 1713-1719,1739-1749, 1753-1758, 1763-1774, 1797-1803, 1807-1846, 1855-1874,1877-1891, 1893-1907, 1912-1925, 1931-1943, 1955-1965, 1976-1990,2032-2043, 2045-2051, 2099-2105, 2131-2138, 2161-2179, 2188-2199,2205-2216, 2219-2227, 2235-2245, 2247-2267, 2277-2288, 2294-2304,2314-2326, 2346-2358, 2365-2377, 2383-2402, 2407-2423, 2437-2450,2454-2473, 2489-2497, 2525-2531, 2557-2570, 2580-2587, 2589-2599,2621-2641, 2647-2653, 2661-2677, 2685-2690, 2697-2717, 2722-2733,2739-2777, 2786-2793, 2801-2808, 2811-2822, 2825-2835, 2838-2845,2859-2871, 2877-2883, 213-344, 954-1080 and 2524-2733 of Seq ID No 279;10-16, 18-23, 28-41, 63-69, 77-91, 101-109, 118-136, 146-153, 155-162,168-179, 192-207, 217-226, 229-235, 239-254, 279-286, 294-307, 313-319,334-341, 344-353, 363-377, 390-396 and 178-328 of Seq ID No 280; 18-42,68-84, 89-95, 100-105, 107-115, 125-135, 154-177, 189-195, 205-228,236-243, 252-259, 279-300, 309-316, 323-331, 340-351, 353-364, 377-402and 85-97 of Seq ID No 281; 4-18, 26-32, 66-76, 100-126, 151-159,178-186, 188-194, 200-210, 241-248, 253-259, 262-279, 284-291, 307-313,315-322, 327-337, 376-386, 399-407, 432-441, 467-473, 487-497, 499-505,543-549, 560-568, 585-593, 598-604, 608-614, 630-642, 647-653, 690-703,717-730, 21-200 and 468-480 of Seq ID No 282; 17-49, 52-58, 62-73,78-97, 100-117, 122-172, 185-190, 193-217, 225-236 and 33-42 of Seq IDNo 283; 7-39, 50-58, 73-89, 96-107, 109-120, 126-142, 152-170, 178-202,205-211, 224-244, 249-259, 261-270, 300-310, 312-325 and 158-169 of SeqID No 284; 4-31, 40-64, 71-82, 85-92, 102-124, 126-139, 147-152,159-173, 176-188, 195-207, 210-216, 234-241, 249-256, 258-276, 279-293,296-302, 310-315, 349-356, 363-378, 380-403, 411-426, 435-441, 448-459,463-476, 488-494 and 201-221 of Seq ID No 285; 5-13, 15-74, 87-104,107-120, 123-129, 136-145, 150-191, 193-206, 227-248, 250-264, 278-302,304-323, 332-378, 384-407, 409-419, 425-457, 462-471, 474-497, 511-545,555-564, 571-578, 585-598, 640-647, 669-675, 682-691, 693-705, 729-743,752-761, 772-780, 786-804, 808-818, 822-846, 858-880, 884-900, 910-939,941-947, 962-971, 973-988, 998-1003, 1007-1027 and 236-259 of Seq ID No286; 4-19, 27-68, 81-111, 121-160 and 60-79 of Seq ID No 287; 4-37,40-46, 52-57, 199-205, 222-229, 236-244, 250-267, 269-282 and 27-197 ofSeq ID No 288; 4-16, 24-30, 32-38, 63-75, 86-92, 98-111, 113-126,160-165, 170-180, 198-204, 227-233, 239-245, 253-273, 308-314, 352-365,382-387, 395-403, 423-429, 472-482, 484-493, 501-507, 518-526, 536-541,543-550, 556-562, 586-600, 626-33, 649-661, 680-688 and 546-559 of SeqID No 289; 16-33, 48-59, 63-71, 77-92, 94-109, 117-124, 139-151,169-181, 184-227, 233-249, 251-261, 263-275, 282-294, 297-321, 326-332,341-355, 383-399 and 258-272 of Seq ID No 290; 11-26, 31-39, 43-52,55-62, 64-70, 80-94, 123-133, 135-141, 172-181, 185-206, 209-218,224-230, 238-244, 251-262, 264-271, 290-301, 306-324, 333-340, 350-357,367-375, 390-397, 434-441, 443-448, 77-226 and 350-429 of Seq ID No 291;4-13, 22-27, 31-45, 50-59, 72-96, 99-114, 131-141, 143-150, 159-176,180-186, 189-198, 208-214, 234-253, 271-287, 294-299, 310-366, 382-390,398-416, 424-443 and 283-305 of Seq ID No 292; 9-26, 30-53, 62-72,86-95, 112-122, 136-145, 153-160, 209-221, 227-237, 241-268, 281-288,291-298, 308-314, 321-328, 336-346, 351-379, 388-397, 409-416, 423-433,443-481, 511-519 and 213-232 of Seq ID No 293; 12-18, 25-31, 38-50,59-67, 71-82, 96-126 and 76-88 of Seq ID No 294; 4-25, 39-44, 64-71,74-88, 100-113, 128-138, 151-162, 164-177, 185-190, 204-213, 233-239,246-254, 281-286, 293-306, 309-318, 333-347, 349-359, 385-398, 404-423,458-465, 477-484, 490-499, 501-533, 554-566, 582-590, 596-616, 624-629,631-639, 654-680, 694-720, 735-743 and 2-100 of Seq ID No 295; 4-16,36-41, 52-75, 98-107, 109-117, 122-128, 133-139, 141-155, 159-165,169-182, 187-193, 195-201, 211-224, 230-236, 247-269, 278-290 and 75-92of Seq ID No 296; 7-21, 25-33, 37-43, 87-94, 103-120, 131-147, 168-174,197-203, 207-212, 227-237, 247-257, 263-271, 279-287, 298-306, 320-325,332-340, 363-374, 379-384, 390-401, 403-414, 428-433, 448-457, 462-475,483-490, 513-519, 525-535, 543-554, 559-566, 571-620, 625-631, 636-642,659-670, 688-706, 708-723, 770-779, 787-793, 796-807, 820-840, 848-854,863-874, 895-905, 912-919, 934-942, 968-975, 983-1000, 1012-1019,1026-1036, 1050-1060, 1064-1070, 1081-1091, 1094-1108, 1112-1118,1140-1152, 1164-1169, 1172-1180, 1187-1192 and 732-748 of Seq ID No 297;23-40, 42-59, 66-73, 78-97, 111-128, 130-141, 157-166, 178-183 and 53-71of Seq ID No 298; 4-27, 38-44, 47-57, 59-85, 99-106, 114-121, 154-166,181-186, 193-198, 238-244, 253-262, 272-278, 287-299, 314-320, 338-350,358-368, 382-388, 407-416, 433-446, 456-461, 463-473 and 86-195 of SeqID No 299; 5-24, 38-59, 64-80, 87-99, 105-126, 134-142, 149-163,165-179, 181-202, 205-220, 227-233, 243-250, 257-263 and 87-245 of SeqID No 300; 5-32, 47-53, 66-79, 81-97, 115-151, 155-174, 183-188,196-210, 215-226, 230-238, 253-258, 263-270, 276-282, 295-301, 304-325,334-344, 360-390, 397-412, 425-432, 434-462, 478-494, 508-526, 539-564,571-579, 347-371 and 375-386 of Seq ID No 301; 4-15, 36-44, 49-56,60-66, 68-82, 84-103, 109-115, 118-141, 147-154, 160-168, 176-185 and26-39 of Seq ID No 302; 7-13, 23-33 and 13-21 of Seq ID No 303; 2-10 ofSeq ID No 304; 4-9, 12-18, 35-42, 49-62 and 6-18 of Seq ID No 305; 19-25and 1-13 of Seq ID No 306; 15-21, 27-45 and 12-25 of Seq ID No 307;14-20 and 1-14 of Seq ID No 308; 4-18 and 13-26 of Seq ID No 309; 8-21and 2-20 of Seq ID No 310; 4-14 and 4-16 of Seq ID No 311; 3-12 of SeqID No 312; 6-14, 6-25, 35-57 and 2-14 of Seq ID No 313; 6-25, 35-57 and17-31 of Seq ID No 314; 14-25, 32-46 and 5-19 of Seq ID No 315; 18-31and 5-16 of Seq ID No 316; 19-24 and 4-26 of Seq ID No 317; 13-21,29-34, 47-58, 61-73 and 36-47 of Seq ID No 318; 4-15 and 5-24 of Seq IDNo 319; 6-18 of Seq ID No 320; 13-20 and 4-13 of Seq ID No 321; 15-23 ofSeq ID No 322; 4-9 and 7-21 of Seq ID No 323; 1-10 of Seq ID No 324;4-14 of Seq ID No 325; 4-17, 35-41, 46-89, 93-98 and 70-88 of Seq ID No326; 1-13 of Seq ID No 327; 4-16, 26-32 and 25-38 of Seq ID No 328;8-15, 23-28 and 4-17 of Seq ID No 329; 4-12 and 1-15 of Seq ID No 330;4-29, 31-42, 52-58 and 6-16 of Seq ID No 331; 4-9, 24-32 and 9-19 of SeqID No 332; 4-12, 18-27 and 5-18 of Seq ID No 333; 4-11, 37-56, 58-92 and18-29 of Seq ID No 334; 8-28 and 20-35 of Seq ID No 335; 4-15 of Seq IDNo 336; 4-23, 27-39, 55-63 and 35-58 of Seq ID No 337; 6-26, 28-54 and28-47 of Seq ID No 338; 4-10, 38-52, 58-82 and 30-49 of Seq ID No 339;4-22, 29-35, 44-50, 53-68, 70-80 and 20-33 of Seq ID No 340; 22-28,30-36 and 18-33 of Seq ID No 341; 4-11, 13-21, 25-30 and 20-30 of Seq IDNo 342; 10-22 and 10-23 of Seq ID No 343; 4-11 and 9-20 of Seq ID No344; 14-25, 32-46 and 6-19 of Seq ID No 345; 5-30 and 14-33 of Seq ID No346; 4-15, 28-35, 46-55, 59-65, 76-84 and 9-24 of Seq ID No 347; 27-33and 5-19 of Seq ID No 348; 5-13 and 8-18 of Seq ID No 349; 9-22, 24-34and 21-40 of Seq ID No 350; 4-17, 35-41, 46-89, 93-98 and 71-89 of SeqID No 351; 4-12, 14-24 and 2-17 of Seq ID No 352; 9-17 and 5-16 of SeqID No 353; 7-41, 48-58, 63-75, 80-89 and 43-53 of Seq ID No 354; 4-22,25-30 and 4-14 of Seq ID No 355; 4-55 and 18-33 of Seq ID No 356;262-280 of Seq ID No 179; 131-146 of Seq ID No 186; 207-224 of Seq ID No188; 27-50, 203-217 and 313-325 of Seq ID No 189; 110-129 of Seq ID No192; 156-179, 174-197, 192-215, 210-233, 228-251 and 246-267 of Seq IDNo 190; 377-400 of Seq ID No 196; 34-43, 234-257 and 350-367 of Seq IDNo 203; 304-327 of Seq ID No 207; 25-48, 43-66 and 61-82 of Seq ID No222; 398-421, 416-439, 434-457, 452-475, 470-493, 488-511, 506-529,524-547, 621-644, 639-664, 707-730, 725-748, 743-766, 761-784, 779-802,797-820, 984-1007, 1002-1025, 1020-1043, 1038-1061, 1056-1079,1074-1097, 1092-1115, 1286-1309, 1304-1327, 1322-1345, 1340-1363,1358-1381, 1376-1399, 1394-1417, 1412-1435, 1430-1453, 1448-1471,1466-1489 and 1484-1507 of Seq ID No 226; 188-211, 206-229, 224-247,242-265, 260-283 and 278-296 of Seq ID No 227; 56-79 and 122-132 of SeqID No 229; 35-46 of Seq ID No 231; 178-201, 196-219, 214-237, 232-255,250-273, 268-291, 379-402, 397-420, 415-438, 433-456, 451-474, 642-65,660-683, 678-701, 696-719, 714-737, 732-755, 750-773, 768-791, 899-922,917-940, 935-958, 1037-1060, 1055-1078, 1073-1096 and 1091-1114 of SeqID No 232; 330-346 of Seq ID No 233; 571-594, 589-612, 607-630, 625-648,643-666 and 661-684 of Seq ID No 242; 188-207 of Seq ID No 244; 61-84,308-331, 326-349, 344-367, 362-385, 380-403 and 398-421 of Seq ID No249; 79-98, 345-366, 844-867, 870-887 and 890-905 of Seq ID No 258;94-109 of Seq ID No 268; 188-207 of Seq ID No 272; 290-306 of Seq ID No276; 826-849 of Seq ID No 277; 228-252, 247-270, 265-288, 283-306,301-324, 955-978, 973-996, 991-1014, 1009-1032, 1027-1050, 1045-1068,2533-2556, 2551-2574, 2569-2592, 2587-2610, 2605-2628 and 2623-2646 ofSeq ID No 279; 86-109 and 104-127 of Seq ID No 288; 546-560 of Seq ID No289; 260-271 of Seq ID No 290; 106-129, 124-147, 142-165, 160-183,178-201 and 375-398 of Seq ID No 291; 284-307 of Seq ID No 292; 362-385of Seq ID No 301.

The present invention also provides a process for producing a H. pylorihyperimmune serum reactive antigen or a fragment thereof according tothe present invention comprising expressing one or more of the nucleicacid molecules according to the present invention in a suitableexpression system.

Moreover, the present invention provides a process for producing a cell,which expresses a H. pylori hyperimmune serum reactive antigen or afragment thereof according to the present invention comprisingtransforming or transfecting a suitable host cell with the vectoraccording to the present invention.

According to the present invention a pharmaceutical composition,especially a vaccine, comprising a hyperimmune serum-reactive antigen ora fragment thereof as defined in the present invention or a nucleic acidmolecule as defined in the present invention is provided.

In a preferred embodiment the pharmaceutical composition furthercomprises an immunostimulatory substance, preferably selected from thegroup comprising polycationic polymers, especially polycationicpeptides, immunostimulatory deoxynucleotides (ODNs), peptides containingat least two LysLeuLys motifs, especially KLKL5KLK, neuroactivecompounds, especially human growth hormone, alumn, Freund's complete orincomplete adjuvants or combinations thereof.

In a more preferred embodiment the immunostimulatory substance is acombination of either a polycationic polymer and immunostimulatorydeoxynucleotides or of a peptide containing at least two LysLeuLysmotifs and immunostimulatory deoxynucleotides.

In a still more preferred embodiment the polycationic polymer is apolycationic peptide, especially polyarginine.

According to the present invention the use of a nucleic acid moleculeaccording to the present invention or a hyperimmune serum-reactiveantigen or fragment thereof according to the present invention for themanufacture of a pharmaceutical preparation, especially for themanufacture of a vaccine against H. pylori infection, is provided.

Also an antibody, or at least an effective part thereof, which binds atleast to a selective part of the hyperimmune serum-reactive antigen or afragment thereof according to the present invention is providedherewith.

In a preferred embodiment the antibody is a monoclonal antibody.

In another preferred embodiment the effective part of the antibodycomprises Fab fragments.

In a further preferred embodiment the antibody is a chimeric antibody.

In a still preferred embodiment the antibody is a humanized antibody.

The present invention also provides a hybridoma cell line, whichproduces an antibody according to the present invention.

Moreover, the present invention provids a method for producing anantibody according to the present invention, characterized by thefollowing steps:

-   -   initiating an immune response in a non-human animal by        administrating an hyperimmune serum-reactive antigen or a        fragment thereof, as defined in the invention, to said animal,    -   removing an antibody containing body fluid from said animal, and    -   producing the antibody by subjecting said antibody containing        body fluid to further purification steps.

Accordingly, the present invention also provides a method for producingan antibody according to the present invention, characterized by thefollowing steps:

-   -   a initiating an immune response in a non-human animal by        administrating an hyperimmune serum-reactive antigen or a        fragment thereof, as defined in the present invention, to said        animal,    -   removing the spleen or spleen cells from said animal,    -   producing hybridoma cells of said spleen or spleen cells,    -   selecting and cloning hybridoma cells specific for said        hyperimmune serum-reactive antigens or a fragment thereof,    -   producing the antibody by cultivation of said cloned hybridoma        cells and optionally further purification steps.

The antibodies provided or produced according to the above methods maybe used for the preparation of a medicament for treating or preventingH. pylori infections.

According to another aspect the present invention provides anantagonist, which binds to a hyperimmune serum-reactive antigen or afragment thereof according to the present invention.

Such an antagonist capable of binding to a hyperimmune serum-reactiveantigen or fragment thereof according to the present invention may beidentified by a method comprising the following steps:

-   -   a) contacting an isolated or immobilized hyperimmune        serum-reactive antigen or a fragment thereof according to the        present invention with a candidate antagonist under conditions        to permit binding of said candidate antagonist to said        hyperimmune serum-reactive antigen or fragment, in the presence        of a component capable of providing a detectable signal in        response to the binding of the candidate antagonist to said        hyperimmune serum reactive antigen or fragment thereof; and    -   b) detecting the presence or absence of a signal generated in        response to the binding of the antagonist to the hyperimmune        serum reactive antigen or the fragment thereof.

An antagonist capable of reducing or inhibiting the interaction activityof a hyperimmune serum-reactive antigen or a fragment thereof accordingto the present invention to its interaction partner may be identified bya method comprising the following steps:

-   -   a) providing a hyperimmune serum reactive antigen or a        hyperimmune fragment thereof according to the present invention,    -   b) providing an interaction partner to said hyperimmune serum        reactive antigen or a fragment thereof, especially an antibody        according to the present invention,    -   c) allowing interaction of said hyperimmune serum reactive        antigen or fragment thereof to said interaction partner to form        an interaction complex,    -   d) providing a candidate antagonist,    -   e) allowing a competition reaction to occur between the        candidate antagonist and the interaction complex,    -   f) determining whether the candidate antagonist inhibits or        reduces the interaction activities of the hyperimmune serum        reactive antigen or the fragment thereof with the interaction        partner.

The hyperimmune serum reactive antigens or fragments thereof accordingto the present invention may be used for the isolation and/orpurification and/or identification of an interaction partner of saidhyperimmune serum reactive antigen or fragment thereof.

The present invention also provides a process for in vitro diagnosing adisease related to expression of a hyperimmune serum-reactive antigen ora fragment thereof according to the present invention comprisingdetermining the presence of a nucleic acid sequence encoding saidhyperimmune serum reactive antigen or fragment thereof according to thepresent invention or the presence of the hyperimmune serum reactiveantigen or fragment thereof according to the present invention.

The present invention also provides a process for in vitro diagnosis ofa bacterial infection, especially a H. pylori infection, comprisinganalyzing for the presence of a nucleic acid sequence encoding saidhyperimmune serum reactive antigen or fragment thereof according to thepresent invention or the presence of the hyperimmune serum reactiveantigen or fragment thereof according to the present invention.

Moreover, the present invention provides the use of a hyperimmune serumreactive antigen or fragment thereof according to the present inventionfor the generation of a peptide binding to said hyperimmune serumreactive antigen or fragment thereof, wherein the peptide is ananticaline.

The present invention also provides the use of a hyperimmuneserum-reactive antigen or fragment thereof according to the presentinvention for the manufacture of a functional nucleic acid, wherein thefunctional nucleic acid is selected from the group comprising aptamersand spiegelmers.

The nucleic acid molecule according to the present invention may also beused for the manufacture of a functional ribonucleic acid, wherein thefunctional ribonucleic acid is selected from the group comprisingribozymes, antisense nucleic acids and siRNA.

The present invention advantageously provides an efficient, relevant andcomprehensive set of isolated nucleic acid molecules and their encodedhyperimmune serum reactive antigens or fragments thereof identified fromH. pylori using an antibody preparation from multiple human plasma poolsand surface expression libraries derived from the genome of H. pylori.Thus, the present invention fulfils a widely felt demand for H. pyloriantigens, vaccines, diagnostics and products useful in procedures forpreparing antibodies and for identifying compounds effective against H.pylori infection.

An effective vaccine should be composed of proteins or polypeptides,which are expressed by all strains and are able to induce high affinity,abundant antibodies against cell surface components of H. pylori. Theantibodies should be IgG1 and/or IgG3 for opsonization, and any IgGsubtype and IgA for neutralisation of adherence and toxin action. Achemically defined vaccine must be definitely superior compared to awhole cell vaccine (attenuated or killed), since components of H.pylori, which cross-react with human tissues or inhibit opsonization canbe eliminated, and the individual proteins inducing protectiveantibodies and/or a protective immune response can be selected.

The approach, which has been employed for the present invention, isbased on the interaction of H. pylori proteins or peptides with theantibodies present in human sera. The antibodies produced against H.pylori by the human immune system and present in human sera areindicative of the in vivo expression of the antigenic proteins and theirimmunogenicity. In addition, the antigenic proteins as identified by thebacterial surface display expression libraries using pools ofpre-selected sera are processed in a second and third round of screeningby individual selected or generated sera. Thus the present inventionsupplies an efficient, relevant, comprehensive set of H. pylori antigensas promising candidates for the development of a pharmaceuticalcomposition, especially a vaccine preventing infection by H. pylori.

In the antigen identification program for identifying a comprehensiveset of antigens according to the present invention, at least twodifferent bacterial surface expression libraries are screened withseveral serum pools or plasma fractions or other pooled antibodycontaining body fluids (antibody pools). The antibody pools are derivedfrom a serum collection, which has been tested against antigeniccompounds of H. pylori, such as whole cell extracts and culturesupernatant proteins. Preferably, 2 distinct serum collections areused: 1. With very stable antibody repertoire: normal adults, clinicallyhealthy people, who are non-carriers and overcame previous encounters orcurrently carriers of H. pylori without acute disease and symptoms, 2.With antibodies induced acutely by the presence of the pathogenicorganism: patients with manifest disease (e.g. H. pylori gastritis,peptic ulcer disease or gastric cancer). Sera have to react withmultiple H. pylori-specific antigens in order to be consideredhyperimmune and therefore relevant in the screening method applied forthe present invention. The antibodies produced against H. pylori by thehuman immune system and present in human sera are indicative of the invivo expression of the antigenic proteins and their immunogenicity.

The expression libraries as used in the present invention should allowexpression of all potential antigens, e.g. derived from all surfaceproteins of H. pylori. Bacterial surface display libraries will berepresented by a recombinant library of a bacterial host displaying a(total) set of expressed peptide sequences of H. pylori on a number ofselected outer membrane proteins (LamB, FhuA) at the bacterial hostmembrane {Georgiou, G., 1997}; {Etz, H. et al., 2001}. One of theadvantages of using recombinant expression libraries is that theidentified hyperimmune serum-reactive antigens may be instantly producedby expression of the coding sequences of the screened and selectedclones expressing the hyperimmune serum-reactive antigens withoutfurther recombinant DNA technology or cloning steps necessary.

The comprehensive set of antigens identified by the described programaccording to the present invention is analysed further by additionalrounds of screening. Therefore individual antibody preparations orantibodies generated against selected peptides, which were identified asimmunogenic are used. According to a preferred embodiment the individualantibody preparations for the second round of screening are derived frompatients who have suffered from infection with H. pylori, especiallyfrom patients who show an antibody titer above a certain minimum level,for example an antibody titer being higher than 80 percentile,preferably higher than 90 percentile, especially higher than 95percentile of the human (patient or healthy individual) sera tested.Using such high titer individual antibody preparations in the secondscreening round allows a very selective identification of thehyperimmune serum-reactive antigens and fragments thereof from H.pylori.

Following the high throughput screening procedure, the selectedantigenic proteins, expressed as recombinant proteins or in vitrotranslated products, in case it can not be expressed in prokaryoticexpression systems, or the identified antigenic peptides (producedsynthetically) are tested in a second screening by a series of ELISA andWestern blotting assays for the assessment of their immunogenicity witha large human serum collection (>50 uninfected, >100 patients sera).

It is important that the individual antibody preparations (which mayalso be the selected serum) allow a selective identification of thehyperimmune serum-reactive antigens from all the promising candidatesfrom the first round. Therefore, preferably at least 10 individualantibody preparations (i.e. antibody preparations (e.g. sera) from atleast 10 different individuals having suffered from an infection to thechosen pathogen) should be used in identifying these antigens in thesecond screening round. It is possible to use also less than 10individual preparations, however, selectivity of the step may not beoptimal with a low number of individual antibody preparations.Therefore, recognition of a given hyperimmune serum-reactive antigen (oran antigenic fragment thereof) by at least 10 individual antibodypreparations, preferably at least 30, especially at least 50 individualantibody preparations confers proper selectivity in the identificationprocess. Hyperimmune serum-reactivity may of course be tested with asmany individual preparations as possible (e.g. with more than 100 oreven with more than 1,000).

Therefore, the relevant portion of the hyperimmune serum-reactiveantibody preparations according to the method of the present inventionshould preferably be at least 10, more preferred at least 30, especiallyat least 50 individual antibody preparations. Alternatively (or incombination) hyperimmune serum-reactive antigens may preferably be alsoidentified with at least 20%, preferably at least 30%, especially atleast 40% of all individual antibody preparations used in the secondscreening round.

According to a preferred embodiment of the present invention, the serafrom which the individual antibody preparations for the second round ofscreening are prepared (or which are used as antibody preparations), areselected by their titer against H. pylori (e.g. against a preparation ofthis pathogen, such as a lysate, cell wall components and recombinantproteins). Preferably, some are selected with a total IgA titer above4,000 U, especially above 6,000 U, and/or an IgG titer above 10,000 U,especially above 12,000 U (U=units, calculated from the OD_(405nm)reading at a given dilution) when the whole organism (total lysate orwhole cells) is used as antigen in the ELISA.

The antibodies produced against Helicobacter by the human immune systemand present in human sera are indicative of the in vivo expression ofthe antigenic proteins and their immunogenicity. The recognition oflinear epitopes by antibodies can be based on sequences as short as 45amino acids. It, however, does not necessarily mean that these shortpeptides are capable of inducing the given antibody in vivo. For thatreason the defined epitopes, polypeptides and proteins are further to betested in animals (mainly in mice) for their capacity to induceantibodies against the selected proteins in vivo.

The preferred antigens are located on the cell surface or secreted, andare therefore accessible extracellularly. Antibodies against cell wallproteins are expected to serve two purposes: to inhibit adhesion and topromote phagocytosis or complement mediated killing. Antibodies againstsecreted proteins are beneficial in neutralisation of their function astoxin or virulence component. It is also known that bacteria communicatewith each other through secreted proteins. Neutralizing antibodiesagainst these proteins will interrupt growth-promoting cross-talkbetween or within Helicobacter species. Bioinformatic analyses (signalsequences, cell wall localisation signals, transmembrane domains) provedto be very useful in assessing cell surface localisation or secretion.The experimental approach includes the isolation of antibodies with thecorresponding epitopes and proteins from human serum, and the generationof immune sera in mice against (poly) peptides selected by the bacterialsurface display screens. These sera are then used in a third round ofscreening as reagents in the following assays: cell surface staining ofHelicobacter grown under different conditions (FACS, microscopy),determination of neutralizing capacity (toxin, adherence), and promotionof opsonization and phagocytosis (in vitro phagocytosis assay).

For that purpose, bacterial E. coli clones are directly injected intomice and immune sera taken and tested in the relevant in vitro assay forfunctional opsonic or neutralizing antibodies. Alternatively, specificantibodies may be purified from human or mouse sera using peptides orproteins as substrate.

It is not clear as to what extend host defence against H. pylori relieson innate or adaptive immunological mechanisms. The mucous membranes andthe gastric acidic environment are formidable barriers against invasionby Helicobacter. However, once the mucous membranes are breached thefirst line of non-adaptive cellular defence begins its co-ordinateaction through complement and phagocytes, especially thepolymorphonuclear leukocytes (PMNs) as indicated by the massiveneutrophil infiltration of the gastric mucosa in response to thepresence of H. pylori. Attachment of H. pylori induces strongpro-inflammatory cytokine release, including TNF-α, IL-1β and IL-8 thatcan mediate a local chemoattractant effect for immuno-effector cells,such as granulocytes {Prinz, C. et al., 2003}; {Sutton, P., 2001}. Thesecells can be regarded as the cornerstones in eliminating invadingbacteria. As H. pylori is thought to be a exclusively extracellularpathogen, the major anti-Helicobacter adaptive response should come fromthe humoral arm of the immune system, and this seems to be in agreementwith the high titers of primarily IgG and IgA antibodies that develop inpatient upon H. pylori infection {Prinz, C. et al., 2003}; {Sutton, P.,2001}. The induction of high titer IgG and sectretory IgA type antibodyresponse may reflect the importance of adaptive mechanisms in the immuneresponse against this organism. In principle, the effect of theseantibodies is mediated through three major mechanisms: promotion ofopsonization, toxin neutralization, and inhibition of adherence. It isbelieved that opsonization is especially important, because of itsrequirement for an effective phagocytosis. For efficient opsonizationthe microbial surface has to be coated with antibodies and complementfactors for recognition by PMNs through receptors for the Fc fragment ofIgG molecules or for activated C3b. After opsonization, the bacteria arephagocytosed and killed. Antibodies bound to specific antigens on thecell surface of bacteria serve as ligands for the attachment to PMNs andto promote phagocytosis. The very same antibodies bound to the adhesinsand other cell surface proteins are expected to neutralize adhesion andprevent colonization.

Inducing high affinity antibodies of the opsonic and neutralizing typeby vaccination helps the innate immune system to eliminate bacteria andtoxins. This makes the method according to the present invention anoptimal tool for the identification of H. pylori antigenic proteins. Theselection of antigens as provided by the present invention is thus wellsuited to identify those that will lead to protection against infectionin an animal model or in humans.

However, there is compelling evidence indicating that antibodies are notrequired for immunisation-induced effective immunity against gastrichelicobacters. Indeed, in vitro studies have demonstrated thatdiminutive fraction of the colonizing H. pylori population might enterepithelial cells and this is in good agreement with the fact that H.pylori generally induces a predominantly T helper 1 (Th1) type immuneresponse, normally associated with invasive bacteria. Gastric T cellsisolated from infected animals and humans produce TNF-α and IFN-γ butnot IL-4, typical for a Th1-biased response. This pro-inflammatory Th1response is clearly not effective against infection {Prinz, C. et al.,2003}; {Sutton, P., 2001}. Current vaccination protocols, such asimmunization with recombinant UreB, can drive the immune response to apolarized Th2 phenotype. Studies with knockout mice demonstrated thatimmunization with urease is possible when the Th2 response is absent.Therefore, vaccine development using new antigens as well as suitableadjuvants that are capable of inducing strong Th1-biased responses maybe beneficial in disease protection caused by H. pylori.

According to the antigen identification method used herein, the presentinvention can surprisingly provide a set of comprehensive novel nucleicacids and novel hyperimmune serum reactive antigens and fragmentsthereof of H. pylori, among other things, as described below. Accordingto one aspect, the invention particularly relates to the nucleotidesequences encoding hyperimmune serum reactive antigens which sequencesare set forth in the Sequence listing Seq ID No 1-178, and thecorresponding encoded amino acid sequences representing hyperimmuneserum reactive antigens are set forth in the Sequence Listing Seq ID No179-356.

In a preferred embodiment of the present invention, a nucleic acidmolecule is provided which exhibits 70% identity over their entirelength to a nucleotide sequence set forth with Seq ID No 3-4, 16, 19-21,28-29, 33-38, 41-42, 44, 48-52, 55, 57-58, 61, 63, 65, 67-68, 72, 74-75,81, 84, 91, 94, 96-97, 101, 105-108, 112, 115-117, 119, 123-178. Mosthighly preferred are nucleic acids that comprise a region that is atleast 80% or at least 85% identical over their entire length to anucleic acid molecule set forth with Seq ID No 3-4, 16, 19-21, 28-29,33-38, 41-42, 44, 48-52, 55, 57-58, 61, 63, 65, 67-68, 72, 74-75, 81,84, 91, 94, 96-97, 101, 105-108, 112, 115-117, 119, 123-178. In thisregard, nucleic acid molecules at least 90%, 91%, 92%, 93%, 94%, 95%, or96% identical over their entire length to the same are particularlypreferred. Furthermore, those with at least 97% are highly preferred,those with at least 98% and at least 99% are particularly highlypreferred, with at least 99% or 99.5% being the more preferred, with100% identity being especially preferred. Moreover, preferredembodiments in this respect are nucleic acids which encode hyperimmuneserum reactive antigens or fragments thereof (polypeptides) which retainsubstantially the same biological function or activity as the maturepolypeptide encoded by said nucleic acids set forth in the Seq ID No3-4, 16, 19-21, 28-29, 33-38, 41-42, 44, 48-52, 55, 57-58, 61, 63, 65,67-68, 72, 74-75, 81, 84, 91, 94, 96-97, 101, 105-108, 112, 115-117,119, 123-178.

Identity, as known in the art and used herein, is the relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,identity also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. Identity canbe readily calculated. While there exist a number of methods to measureidentity between two polynucleotides or two polypeptide sequences, theterm is well known to skilled artisans (e.g. Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987). Preferredmethods to determine identity are designed to give the largest matchbetween the sequences tested. Methods to determine identity are codifiedin computer programs. Preferred computer program methods to determineidentity between two sequences include, but are not limited to, GCGprogram package {Devereux, J. et al., 1984}, BLASTP, BLASTN, and FASTA{Altschul, S. et al., 1990}.

According to another aspect of the invention, nucleic acid molecules areprovided which exhibit at least 96% identity to the nucleic acidsequence set forth with Seq ID No 8-10, 13-15, 17-18, 24, 27, 32, 39-40,45-47, 56, 59, 62, 69-70, 73, 77, 79, 82, 85-86, 88, 90, 103, 109-110,114, 121.

According to a further aspect of the present invention, nucleic acidmolecules are provided which are identical to the nucleic acid sequencesset forth with Seq ID No 5, 7, 30-31, 53, 60, 66, 76, 83, 87, 92, 99,120.

The nucleic acid molecules according to the present invention can as asecond alternative also be a nucleic acid molecule which is at leastessentially complementary to the nucleic acid described as the firstalternative above. As used herein complementary means that a nucleicacid strand is base pairing via Watson-Crick base pairing with a secondnucleic acid strand. Essentially complementary as used herein means thatthe base pairing is not occurring for all of the bases of the respectivestrands but leaves a certain number or percentage of the bases unpairedor wrongly paired. The percentage of correctly pairing bases ispreferably at least 70%, more preferably 80%, even more preferably 90%and most preferably any percentage higher than 90%. It is to be notedthat a percentage of 70% matching bases is considered as homology andthe hybridization having this extent of matching base pairs isconsidered as stringent. Hybridization conditions for this kind ofstringent hybridization may be taken from Current Protocols in MolecularBiology (John Wiley and Sons, Inc, 1987). More particularly, thehybridization conditions can be as follows:

-   -   Hybridization performed e.g. in 5×SSPE, 5×Denhardt's reagent,        0.1% SDS, 100 g/mL sheared DNA at 68° C.    -   Moderate stringency wash in 0.2×SSC, 0.1% SDS at 42° C.    -   High stringency wash in 0.1×SSC, 0.1% SDS at 68° C.

Genomic DNA with a GC content of 50% has an approximate TM of 96° C. For1% mismatch, the TM is reduced by approximately 1° C.

In addition, any of the further hybridization conditions describedherein is in principle applicable as well.

All nucleic acid sequence molecules which encode the same polypeptidemolecule as those identified by the present invention are encompassed byany disclosure of a given coding sequence, since the degeneracy of thegenetic code is directly applicable to unambiguously determine allpossible nucleic acid molecules which encode a given polypeptidemolecule, even if the number of such degenerated nucleic acid moleculesmay be high. This is also applicable for fragments of a givenpolypeptide, as long as the fragments encode a polypeptide beingsuitable to be used in a vaccination connection, e.g. as an active orpassive vaccine.

The nucleic acid molecule according to the present invention can as athird alternative also be a nucleic acid which comprises a stretch of atleast 15 bases of the nucleic acid molecule according to the first andsecond alternative of the nucleic acid molecules according to thepresent invention as outlined above. Preferably, the bases form acontiguous stretch of bases. However, it is also within the scope of thepresent invention that the stretch consists of two or more moieties,which are separated by a number of bases.

The present nucleic acids may preferably consist of at least 20, evenmore preferred at least 30, especially at least 50 contiguous bases fromthe sequences disclosed herein. The suitable length may easily beoptimized due to the planned area of use (e.g. as (PCR) primers, probes,capture molecules (e.g. on a (DNA) chip), etc.). Preferred nucleic acidmolecules contain at least a contiguous 15 base portion of one or moreof the predicted immunogenic amino acid sequences listed in tables 1 and2, especially the sequences of table 2 with scores of more than 10,preferably more than 20, especially with a score of more than 25.Specifically preferred are nucleic acids containing a contiguous portionof a DNA sequence of any sequence in the sequence protocol of thepresent application which shows 1 or more, preferably more than 2,especially more than 5, non-identical nucleic acid residues compared tothe published Helicobacter pylori strain 26695 and J99 genomes (Nature,388: 539-547 (1997), 4658-4663; GenBank accession AE000511 and Nature,397: 176-180 (1999), GenBank accession AE001439) and/or any otherpublished H. pylori genome sequence or parts thereof. Specificallypreferred non-identical nucleic acid residues are residues, which leadto a non-identical amino acid residue. Preferably, the nucleic acidsequences encode for polypeptides having at least 1, preferably at least2, preferably at least three different amino acid residues compared tothe published H. pylori counterparts mentioned above. Also such isolatedpolypeptides, being fragments of the proteins (or the whole protein)mentioned herein e.g. in the sequence listing, having at least 6, 7, or8 amino acid residues and being encoded by these nucleic acids arepreferred.

The nucleic acid molecule according to the present invention can as afourth alternative also be a nucleic acid molecule which anneals understringent hybridisation conditions to any of the nucleic acids of thepresent invention according to the above outlined first, second, andthird alternative. Stringent hybridisation conditions are typicallythose described herein.

Finally, the nucleic acid molecule according to the present inventioncan as a fifth alternative also be a nucleic acid molecule which, butfor the degeneracy of the genetic code, would hybridise to any of thenucleic acid molecules according to any nucleic acid molecule of thepresent invention according to the first, second, third, and fourthalternative as outlined above. This kind of nucleic acid molecule refersto the fact that preferably the nucleic acids according to the presentinvention code for the hyperimmune serum reactive antigens or fragmentsthereof according to the present invention. This kind of nucleic acidmolecule is particularly useful in the detection of a nucleic acidmolecule according to the present invention and thus the diagnosis ofthe respective microorganisms such as H. pylori and any disease ordiseased condition where this kind of microorganim is involved.Preferably, the hybridisation would occur or be preformed understringent conditions as described in connection with the fourthalternative described above.

Nucleic acid molecule as used herein generally refers to any ribonucleicacid molecule or deoxyribonucleic acid molecule, which may be unmodifiedRNA or DNA or modified RNA or DNA. Thus, for instance, nucleic acidmolecule as used herein refers to, among other, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded RNA, and RNA that is a mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded, or a mixture of single- and double-stranded regions. Inaddition, nucleic acid molecule as used herein refers to triple-strandedregions comprising RNA or DNA or both RNA and DNA. The strands in suchregions may be from the same molecule or from different molecules. Theregions may include all of one or more of the molecules, but moretypically involve only a region of some of the molecules. One of themolecules of a triple-helical region often is an oligonucleotide. Asused herein, the term nucleic acid molecule includes DNAs or RNAs asdescribed above that contain one or more modified bases. Thus, DNAs orRNAs with backbones modified for stability or for other reasons are“nucleic acid molecule” as that term is intended herein. Moreover, DNAsor RNAs comprising unusual bases, such as inosine, or modified bases,such as tritylated bases, to name just two examples, are nucleic acidmolecule as the term is used herein. It will be appreciated that a greatvariety of modifications have been made to DNA and RNA that serve manyuseful purposes known to those of skill in the art. The term nucleicacid molecule as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of nucleic acid molecule,as well as the chemical forms of DNA and RNA characteristic of virusesand cells, including simple and complex cells, inter alia. The termnucleic acid molecule also embraces short nucleic acid molecules oftenreferred to as oligonucleotide(s). “Polynucleotide” and “nucleic acid”or “nucleic acid molecule” are often used interchangeably herein.

Nucleic acid molecules provided in the present invention also encompassnumerous unique fragments, both longer and shorter than the nucleic acidmolecule sequences set forth in the sequencing listing of the H. pyloricoding regions, which can be generated by standard cloning methods. Tobe unique, a fragment must be of sufficient size to distinguish it fromother known nucleic acid sequences, most readily determined by comparingany selected H. pylori fragment to the nucleotide sequences in computerdatabases such as GenBank.

Additionally, modifications can be made to the nucleic acid moleculesand polypeptides that are encompassed by the present invention. Forexample, nucleotide substitutions can be made which do not affect thepolypeptide encoded by the nucleic acid, and thus any nucleic acidmolecule which encodes a hyperimmune serum reactive antigen or fragmentsthereof is encompassed by the present invention.

Furthermore, any of the nucleic acid molecules encoding hyperimmuneserum reactive antigens or fragments thereof provided by the presentinvention can be functionally linked, using standard techniques such asstandard cloning techniques, to any desired regulatory sequences,whether a H. pylori regulatory sequence or a heterologous regulatorysequence, heterologous leader sequence, heterologous marker sequence ora heterologous coding sequence to create a fusion protein.

Nucleic acid molecules of the present invention may be in the form ofRNA, such as mRNA or cRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or produced bychemical synthetic techniques or by a combination thereof. The DNA maybe triple-stranded, double-stranded or single-stranded. Single-strandedDNA may be the coding strand, also known as the sense strand, or it maybe the non-coding strand, also referred to as the anti-sense strand.

The present invention further relates to variants of the herein abovedescribed nucleic acid molecules which encode fragments, analogs andderivatives of the hyperimmune serum reactive antigens and fragmentsthereof having a deducted H. pylori amino acid sequence set forth in theSequence Listing. A variant of the nucleic acid molecule may be anaturally occurring variant such as a naturally occurring allelicvariant, or it may be a variant that is not known to occur naturally.Such non-naturally occurring variants of the nucleic acid molecule maybe made by mutagenesis techniques, including those applied to nucleicacid molecules, cells or organisms.

Among variants in this regard are variants that differ from theaforementioned nucleic acid molecules by nucleotide substitutions,deletions or additions. The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingor non-coding regions or both. Alterations in the coding regions mayproduce conservative or non-conservative amino acid substitutions,deletions or additions. Preferred are nucleic acid molecules encoding avariant, analog, derivative or fragment, or a variant, analogue orderivative of a fragment, which have a H. pylori sequence as set forthin the Sequence Listing, in which several, a few, 5 to 10, 1 to 5, 1 to3, 2, 1 or no amino acid(s) is substituted, deleted or added, in anycombination. Especially preferred among these are silent substitutions,additions and deletions, which do not alter the properties andactivities of the H. pylori polypeptides set forth in the SequenceListing. Also especially preferred in this regard are conservativesubstitutions.

The peptides and fragments according to the present invention alsoinclude modified epitopes wherein preferably one or two of the aminoacids of a given epitope are modified or replaced according to the rulesdisclosed in e.g. {Tourdot, S. et al., 2000}, as well as the nucleicacid sequences encoding such modified epitopes.

It is clear that also epitopes derived from the present epitopes byamino acid exchanges improving, conserving or at least not significantlyimpeding the T cell activating capability of the epitopes are covered bythe epitopes according to the present invention. Therefore the presentepitopes also cover epitopes, which do not contain the original sequenceas derived from H. pylori, but trigger the same or preferably animproved T cell response. These epitope are referred to as“heteroclitic”; they need to have a similar or preferably greateraffinity to MHC/HLA molecules, and the need the ability to stimulate theT cell receptors (TCR) directed to the original epitope in a similar orpreferably stronger manner.

Heteroclitic epitopes can be obtained by rational design i.e. takinginto account the contribution of individual residues to binding toMHC/HLA as for instance described by {Rammensee, H. et al., 1999},combined with a systematic exchange of residues potentially interactingwith the TCR and testing the resulting sequences with T cells directedagainst the original epitope. Such a design is possible for a skilledman in the art without much experimentation.

Another possibility includes the screening of peptide libraries with Tcells directed against the original epitope. A preferred way is thepositional scanning of synthetic peptide libraries. Such approaches havebeen described in detail for instance by {Hemmer, B. et al., 1999} andthe references given therein.

As an alternative to epitopes represented by the present derived aminoacid sequences or heteroclitic epitopes, also substances mimicking theseepitopes e.g. “peptidemimetica” or “retro-inverso-peptides” can beapplied.

Another aspect of the design of improved epitopes is their formulationor modification with substances increasing their capacity to stimulate Tcells. These include T helper cell epitopes, lipids or liposomes orpreferred modifications as described in WO 01/78767.

Another way to increase the T cell stimulating capacity of epitopes istheir formulation with immune stimulating substances for instancecytokines or chemokines like interleukin-2, -7, -12, -18, class I and IIinterferons (IFN), especially IFN-gamma, GM-CSF, TNF-alpha, flt3-ligandand others.

As discussed additionally herein regarding nucleic acid molecule assaysof the invention, for instance, nucleic acid molecules of the inventionas discussed above, may be used as a hybridization probe for RNA, cDNAand genomic DNA to isolate full-length cDNAs and genomic clones encodingpolypeptides of the present invention and to isolate cDNA and genomicclones of other genes that have a high sequence similarity to thenucleic acid molecules of the present invention. Such probes generallywill comprise at least 15 bases. Preferably, such probes will have atleast 20, at least 25 or at least 30 bases, and may have at least 50bases. Particularly preferred probes will have at least 30 bases, andwill have 50 bases or less, such as 30, 35, 40, 45, or 50 bases.

For example, the coding region of a nucleic acid molecule of the presentinvention may be isolated by screening a relevant library using theknown DNA sequence to synthesize an oligonucleotide probe. A labeledoligonucleotide having a sequence complementary to that of a gene of thepresent invention is then used to screen a library of cDNA, genomic DNAor mRNA to determine to which members of the library the probehybridizes.

The nucleic acid molecules and polypeptides of the present invention maybe employed as reagents and materials for development of treatments ofand diagnostics for disease, particularly human disease, as furtherdiscussed herein relating to nucleic acid molecule assays, inter alia.

The nucleic acid molecules of the present invention that areoligonucleotides can be used in the processes herein as described, butpreferably for PCR, to determine whether or not the H. pylori genesidentified herein in whole or in part are present and/or transcribed ininfected tissue such as blood. It is recognized that such sequences willalso have utility in diagnosis of the stage of infection and type ofinfection the pathogen has attained. For this and other purposes thearrays comprising at least one of the nucleic acids according to thepresent invention as described herein, may be used.

The nucleic acid molecules according to the present invention may beused for the detection of nucleic acid molecules and organisms orsamples containing these nucleic acids. Preferably such detection is fordiagnosis, more preferable for the diagnosis of a disease related orlinked to the present or abundance of H. pylori.

Eukaryotes (herein also “individual(s)”), particularly mammals, andespecially humans, infected with H. pylori may be identifiable bydetecting any of the nucleic acid molecules according to the presentinvention detected at the DNA level by a variety of techniques.Preferred nucleic acid molecules candidates for distinguishing a H.pylori from other organisms can be obtained.

The invention provides a process for diagnosing disease, arising frominfection with H. pylori, comprising determining from a sample isolatedor derived from an individual an increased level of expression of anucleic acid molecule having the sequence of a nucleic acid molecule setforth in the Sequence Listing. Expression of nucleic acid molecules canbe measured using any one of the methods well known in the art for thequantitation of nucleic acid molecules, such as, for example, PCR,RT-PCR, Rnase protection, Northern blotting, other hybridisation methodsand the arrays described herein.

Isolated as used herein means separated “by the hand of man” from itsnatural state; i.e., that, if it occurs in nature, it has been changedor removed from its original environment, or both. For example, anaturally occurring nucleic acid molecule or a polypeptide naturallypresent in a living organism in its natural state is not “isolated,” butthe same nucleic acid molecule or polypeptide separated from thecoexiting materials of its natural state is “isolated”, as the term isemployed herein. As part of or following isolation, such nucleic acidmolecules can be joined to other nucleic acid molecules, such as DNAs,for mutagenesis, to form fusion proteins, and for propagation orexpression in a host, for instance. The isolated nucleic acid molecules,alone or joined to other nucleic acid molecules such as vectors, can beintroduced into host cells, in culture or in whole organisms. Introducedinto host cells in culture or in whole organisms, such DNAs still wouldbe isolated, as the term is used herein, because they would not be intheir naturally occurring form or environment. Similarly, the nucleicacid molecules and polypeptides may occur in a composition, such as amedia formulations, solutions for introduction of nucleic acid moleculesor polypeptides, for example, into cells, compositions or solutions forchemical or enzymatic reactions, for instance, which are not naturallyoccurring compositions, and, therein remain isolated nucleic acidmolecules or polypeptides within the meaning of that term as it isemployed herein.

The nucleic acids according to the present invention may be chemicallysynthesized. Alternatively, the nucleic acids can be isolated from H.pylori by methods known to the one skilled in the art.

According to another aspect of the present invention, a comprehensiveset of novel hyperimmune serum reactive antigens and fragments thereofare provided by using the herein described antigen identificationmethod. In a preferred embodiment of the invention, a hyperimmuneserum-reactive antigen comprising an amino acid sequence being encodedby any one of the nucleic acids molecules herein described and fragmentsthereof are provided. In another preferred embodiment of the invention anovel set of hyperimmune serum-reactive antigens which comprises aminoacid sequences selected from a group consisting of the polypeptidesequences as represented in Seq ID No 181-182, 194, 197-199, 206-207,211-216, 219-220, 222, 226-230, 233, 235-236, 239, 241, 243, 245-246,250, 252-253, 259, 262, 269, 272, 274-275, 279, 283-286, 290, 293-295,297, 301-356 and fragments thereof are provided. In a further preferredembodiment of the invention hyperimmune serum-reactive antigens, whichcomprise amino acid sequences selected from a group consisting of thepolypeptide sequences as represented in Seq ID No 186-188, 191-193,195-196, 202, 205, 210, 217-218, 223-225, 234, 237, 240, 247-248, 251,255, 257, 260, 263-264, 266, 268, 281, 287-288, 292, 299 and fragmentsthereof are provided. In a still preferred embodiment of the inventionhyperimmune serum-reactive antigens which comprise amino acid sequencesselected from a group consisting of the polypeptide sequences asrepresented in Seq ID No 183, 185, 208-209, 231, 238, 244, 254, 261,265, 270, 277, 298 and fragments thereof are provided.

The hyperimmune serum reactive antigens and fragments thereof asprovided in the invention include any polypeptide set forth in theSequence Listing as well as polypeptides which have at least 70%identity to a polypeptide set forth in the Sequence Listing, preferablyat least 80% or 85% identity to a polypeptide set forth in the SequenceListing, and more preferably at least 90% similarity (more preferably atleast 90% identity) to a polypeptide set forth in the Sequence Listingand still more preferably at least 95%, 96%, 97%, 98%, 99% or 99.5%similarity (still more preferably at least 95%, 96%, 97%, 98%, 99%, or99.5% identity) to a polypeptide set forth in the Sequence Listing andalso include portions of such polypeptides with such portion of thepolypeptide generally containing at least 4 amino acids and morepreferably at least 8, still more preferably at least 30, still morepreferably at least 50 amino acids, such as 4, 8, 10, 20, 30, 35, 40, 45or 50 amino acids.

The invention also relates to fragments, analogs, and derivatives ofthese hyperimmune serum reactive antigens and fragments thereof. Theterms “fragment”, “derivative” and “analog” when referring to an antigenwhose amino acid sequence is set forth in the Sequence Listing, means apolypeptide which retains essentially the same or a similar biologicalfunction or activity as such hyperimmune serum reactive antigen andfragment thereof.

The fragment, derivative or analog of a hyperimmune serum reactiveantigen and fragment thereof may be 1) one in which one or more of theamino acid residues are substituted with a conserved or non-conservedamino acid residue (preferably a conserved amino acid residue) and suchsubstituted amino acid residue may or may not be one encoded by thegenetic code, or 2) one in which one or more of the amino acid residuesincludes a substituent group, or 3) one in which the mature hyperimmuneserum reactive antigen or fragment thereof is fused with anothercompound, such as a compound to increase the half-life of thehyperimmune serum reactive antigen and fragment thereof (for example,polyethylene glycol), or 4) one in which the additional amino acids arefused to the mature hyperimmune serum reactive antigen or fragmentthereof, such as a leader or secretory sequence or a sequence which isemployed for purification of the mature hyperimmune serum reactiveantigen or fragment thereof or a proprotein sequence. Such fragments,derivatives and analogs are deemed to be within the scope of thoseskilled in the art from the teachings herein.

Among the particularly preferred embodiments of the invention in thisregard are the hyperimmune serum reactive antigens set forth in theSequence Listing, variants, analogs, derivatives and fragments thereof,and variants, analogs and derivatives of fragments. Additionally, fusionpolypeptides comprising such hyperimmune serum reactive antigens,variants, analogs, derivatives and fragments thereof, and variants,analogs and derivatives of the fragments are also encompassed by thepresent invention. Such fusion polypeptides and proteins, as well asnucleic acid molecules encoding them, can readily be made using standardtechniques, including standard recombinant techniques for producing andexpression of a recombinant polynucleic acid encoding a fusion protein.

Among preferred variants are those that vary from a reference byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid in a polypeptide by another amino acid oflike characteristics. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe and Tyr.

Further particularly preferred in this regard are variants, analogs,derivatives and fragments, and variants, analogs and derivatives of thefragments, having the amino acid sequence of any polypeptide set forthin the Sequence Listing, in which several, a few, 5 to 10, 1 to 5, 1 to3, 2, 1 or no amino acid residues are substituted, deleted or added, inany combination. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the polypeptide of the present invention.Also especially preferred in this regard are conservative substitutions.Most highly preferred are polypeptides having an amino acid sequence setforth in the Sequence Listing without substitutions.

The hyperimmune serum reactive antigens and fragments thereof of thepresent invention are preferably provided in an isolated form, andpreferably are purified to homogeneity.

Also among preferred embodiments of the present invention arepolypeptides comprising fragments of the polypeptides having the aminoacid sequence set forth in the Sequence Listing, and fragments ofvariants and derivatives of the polypeptides set forth in the SequenceListing.

In this regard a fragment is a polypeptide having an amino acid sequencethat entirely is the same as part but not all of the amino acid sequenceof the afore mentioned hyperimmune serum reactive antigen and fragmentthereof, and variants or derivative, analogs, fragments thereof. Suchfragments may be “free-standing”, i.e., not part of or fused to otheramino acids or polypeptides, or they may be comprised within a largerpolypeptide of which they form a part or region. Also preferred in thisaspect of the invention are fragments characterised by structural orfunctional attributes of the polypeptide of the present invention, i.e.fragments that comprise alpha-helix and alpha-helix forming regions,beta-sheet and beta-sheet forming regions, turn and turn-formingregions, coil and coil-forming regions, hydrophilic regions, hydrophobicregions, alpha amphipathic regions, beta-amphipathic regions, flexibleregions, surface-forming regions, substrate binding regions, and highantigenic index regions of the polypeptide of the present invention, andcombinations of such fragments. Preferred regions are those that mediateactivities of the hyperimmune serum reactive antigens and fragmentsthereof of the present invention. Most highly preferred in this regardare fragments that have a chemical, biological or other activity of thehyperimmune serum reactive antigen and fragments thereof of the presentinvention, including those with a similar activity or an improvedactivity, or with a decreased undesirable activity. Particularlypreferred are fragments comprising receptors or domains of enzymes thatconfer a function essential for viability of H. pylori or the ability tocause disease in humans. Further preferred polypeptide fragments arethose that comprise or contain antigenic or immunogenic determinants inan animal, especially in a human.

An antigenic fragment is defined as a fragment of the identifiedantigen, which is for itself antigenic or may be made antigenic whenprovided as a hapten. Therefore, also antigens or antigenic fragmentsshowing one or (for longer fragments) only a few amino acid exchangesare enabled with the present invention, provided that the antigeniccapacities of such fragments with amino acid exchanges are not severelydeteriorated on the exchange(s), i.e., suited for eliciting anappropriate immune response in an individual vaccinated with thisantigen and identified by individual antibody preparations fromindividual sera.

Preferred examples of such fragments of a hyperimmune serum-reactiveantigen are selected from the group consisting of peptides comprisingamino acid sequences of column “predicted immunogenic aa”, and “Locationof identified immunogenic region” of Table 1, the serum reactive epitopeof Table 3: especially peptides comprising amino acids 63-91, 95-101,110-116, 134-148, 150-156, 158-164, 188-193, 197-209, 226-241, 247-254,291-297, 312-319, 338-346, 351-358, 366-378, 404-410, 420-438, 448-454,465-473, 482-488, 490-498, 503-510, 512-519, 531-543, 547-554, 568-575,589-604, 610-631 and 239-308 of Seq ID No 179; 16-29, 35-47, 50-68,70-79, 91-101, 143-149, 158-163, 185-191, 196-206, 215-224, 230-237,244-251, 258-278, 290-311, 319-325, 338-351, 365-385, 396-429, 445-454,458-466, 491-499, 501-521, 17-79 and 218-233 of Seq ID No 180; 4-10,16-41, 46-66, 77-84, 91-97, 102-118, 125-144, 187-200, 202-214, 245-253,255-261, 286-295, 300-330, 335-342, 350-361, 363-381, 385-392, 396-416,435-450 and 460-470 of Seq ID No 181; 11-19, 27-48, 52-59, 77-82,84-107, 118-125, 127-154, 178-183, 192-209, 215-221, 286-295, 302-313,350-357, 402-415, 417-431, 453-463, 465-493 and 313-331 of Seq ID No182; 19-26, 30-43, 47-55, 63-68, 72-80, 97-104, 107-119, 129-146,160-175, 194-216, 231-251, 254-260 and 26-43 of Seq ID No 183; 7-13,29-37, 6-81, 110-120, 123-131, 135-152, 230-249, 254-260, 284-290,292-299, 317-326, 329-336, 403-444, 452-458, 466-477, 490-498, 510-519,541-550, 557-566 and 533-567 of Seq ID No 184; 5-47, 71-77, 79-86,89-95, 120-126, 137-144, 176-181, 184-196, 202-208, 211-232, 236-282,301-313, 317-325, 341-347, 353-384, 394-400, 412-433, 436-443 and 59-75of Seq ID No 185; 4-18, 22-38, 59-69, 106-112, 116-130, 138-149,156-170, 175-197, 200-214, 216-223, 233-244, 255-261, 266-276, 279-286,325-333, 342-348, 366-399, 402-420, 429-441, 1-104 and 130-147 of Seq IDNo 186; 50-58, 69-95, 97-113, 131-136, 157-163, 170-175, 188-212,220-226, 254-259, 265-277, 283-289, 297-308, 311-318, 347-358, 360-369,378-401, 416-421, 440-450, 454-462, 470-476, 493-502, 506-514, 536-567,585-590, 598-607, 613-618, 653-659 and 35-46 of Seq ID No 187; 16-29,32-60, 65-87, 89-123, 128-134, 137-158, 162-173, 178-196, 210-216,218-228 and 206-225 of Seq ID No 188; 10-20, 26-35, 51-64, 86-91,94-100, 113-122, 154-160, 185-191, 193-201, 211-217, 225-230, 237-246,251-257, 298-304, 306-312, 316-328, 340-348, 357-389, 391-397, 415-421,449-456, 458-471, 488-495, 502-511, 24-55 and 236-341 of Seq ID No 189;5-22, 41-51, 87-93, 114-122, 127-136, 150-156, 158-166, 223-233,245-263, 291-296, 9-126 and 127-285 of Seq ID No 190; 30-43, 46-56,61-70, 72-83, 85-93, 103-113, 119-125, 151-166, 179-191, 212-218,225-231, 236-243, 262-267, 291-307, 331-44, 349-355, 366-372, 380-386,414-422, 428-447, 459-464, 469-478, 507-519, 525-544, 563-569, 576-590,620-626, 633-643, 654-659, 665-671, 684-707, 717-723, 725-733, 747-779,782-801 and 347-361 of Seq ID No 191; 4-12, 14-26, 37-80, 107-115,133-139, 144-150, 154-165, 173-180, 191-199, 205-211, 221-231, 237-244,254-284, 307-340, 342-353, 360-368, 370-380, 479-493, 495-503, 509-522,525-536, 539-547, 554-560, 565-573, 578-583, 7-23 and 465-479 of Seq IDNo 192; 4-17, 47-55, 76-83, 85-100, 104-112, 117-123, 126-135, 142-148,156-167, 174-182, 267-273 and 258-283 of Seq ID No 193; 8-32, 36-42,65-88, 102-108, 112-140, 147-163, 170-179, 183-193 and 117-124 of Seq IDNo 194; 12-18, 45-50, 62-77, 82-95, 99-113, 115-123, 125-147, 155-177,187-209, 211-223, 244-253, 259-270, 278-297, 302-307, 311-318, 329-334,350-356, 359-365, 390-400, 402-413 and 333-350 of Seq ID No 195; 4-13,15-27, 30-46, 53-58, 68-74, 82-95, 115-126, 134-139, 148-153, 159-176,182-199, 201-217, 220-225, 227-235, 237-248, 253-266, 300-315, 322-336,390-396, 412-426, 438-445, 448-459, 477-484, 502-508, 515-527, 529-537,553-568, 643-651, 658-667, 690-703 and 376-400 of Seq ID No 196; 4-10,24-32, 38-55, 59-67, 70-77, 80-87, 89-97, 123-129, 134-151, 166-172,178-189, 191-216, 218-235, 245-259, 271-315, 326-339, 341-360 and 73-94of Seq ID No 197; 13-25, 31-38, 43-57, 79-85, 92-99, 106-112, 117-128,130-139, 146-158, 160-175, 194-204, 211-222, 225-232, 234-242, 263-270,278-292, 299-320, 322-333 and 240-256 of Seq ID No 198; 4-17, 55-63,66-101, 109-131, 135-143, 145-151, 155-161, 164-170, 177-185, 192-198,213-218, 223-238, 246-256, 258-268, 273-283, 309-314, 322-328 and195-221 of Seq ID No 199; 13-24, 31-39, 41-50, 63-69, 90-96, 104-109,116-141, 148-153, 161-167, 173-178, 190-209, 253-258, 265-272, 279-289,295-312, 317-343, 355-366, 376-389, 400-407, 430-451, 453-464, 466-472,487-493, 499-505, 523-538, 554-559, 568-579, 584-601 and 344-363 of SeqID No 200; 5-22, 30-36, 53-59, 61-70, 82-92, 99-106, 120-131, 135-148,154-167, 169-183, 187-199, 204-212, 231-247 and 111-249 of Seq ID No201; 17-36, 40-66, 71-144, 148-171, 173-191, 199-214, 220-252, 265-272,278-288, 298-333, 342-385 and 287-307 of Seq ID No 202; 4-16, 22-28,30-36, 42-48, 95-116, 154-162, 164-174, 239-252, 258-263, 273-285,306-313, 323-333, 341-357, 363-369, 372-379, 395-401, 430-436, 438-453,464-480, 33-44, 233-258 and 349-369 of Seq ID No 203; 4-21, 30-37,46-53, 59-68, 80-92, 98-104, 118-143, 150-160, 165-185, 187-200,204-211, 224-236, 241-246, 252-258, 271-280, 288-294, 311-320, 335-341and 191-350 of Seq ID No 204; 4-16, 37-59, 64-70, 79-87, 93-102,107-127, 143-165, 172-188, 197-204, 207-218, 221-227, 242-248, 258-277,289-296, 298-316, 332-338, 344-365, 367-373, 375-382, 400-408, 415-425,438-446 and 235-250 of Seq ID No 205; 4-37, 39-66, 84-98, 101-127,140-149, 157-163, 166-172, 175-182, 184-193, 203-208, 215-232, 234-247,250-299, 303-345 and 183-204 of Seq ID No 206; 10-20, 41-61, 73-87,112-141, 176-192, 194-201, 205-222, 230-237, 257-264, 276-282, 284-310,312-318, 330-337, 349-357 and 304-328 of Seq ID No 207; 4-31, 42-103,105-113, 121-153, 160-181, 188-196, 210-226, 231-264, 272-287, 297-304,328-336 and 304-318 of Seq ID No 208; 21-43, 46-52, 54-70, 72-79,94-107, 133-141, 160-166, 217-253, 311-317, 359-365, 374-381, 390-395,434-440, 488-494, 497-502, 511-522, 554-563, 565-574, 577-585, 591-598,601-606, 617-625, 633-643, 658-664, 676-682, 694-702, 710-719, 754-760,782-788, 802-808, 916-921, 942-948, 955-964, 973-979, 992-998,1006-1011, 1016-1023, 1030-1038, 1046-1053, 1059-1066, 1088-1098,1119-1126, 1129-1135, 1156-1171, 1173-1181, 1202-1210, 1255-1261,1268-1280, 1295-1310, 1312-1320, 1375-1381, 1406-1417, 1450-1471,1478-1492, 1498-1506, 1569-1578, 1603-1608, 1611-1624, 1648-1655,1663-1670, 1680-1698, 1702-1707, 1713-1719, 1737-1742, 1747-1753,1762-1769, 1771-1785, 1790-1804, 1811-1818, 1830-1836, 1838-1852,1874-1886, 1893-1899, 1902-1909, 1942-1948, 1952-1962, 1980-1986,2001-2017, 2020-2028, 2042-2050, 2052-2068, 2074-2079, 2083-2095,2107-2113, 2147-2155, 2177-2194, 2203-2211, 2236-2241, 2251-2258,2267-2274, 2285-2292, 2314-2328, 2330-2340, 2358-2365, 2390-2401,2408-2418, 2432-2453, 2463-2476, 2486-2507, 2528-2537, 2540-2548,2552-2558, 2568-2576, 2596-2601, 2610-2622, 2629-2638, 2653-2669,2718-2727, 2749-2767, 2777-2784, 2789-2795, 2806-2815, 2817-2824,2835-2843, 2847-2854, 2860-2881, 511-523, 612-630 and 1790-1803 of SeqID No 209; 4-54, 61-68, 72-82, 86-93, 100-108, 115-130, 147-154,187-194, 196-207, 224-229, 236-251, 275-287 and 96-109 of Seq ID No 210;31-39, 62-69, 91-101, 158-172, 175-180, 186-193, 201-208, 210-223,243-250, 273-286, 293-299, 319-325, 343-354, 356-365, 368-384, 414-435,471-491, 512-518, 550-556, 567-581, 584-589, 633-639, 680-692, 697-708,716-721, 747-754, 779-786, 810-816 and 366-503 of Seq ID No 211; 5-20,22-48, 57-65, 96-101, 111-122, 130-145, 154-164, 170-181, 193-199,201-216, 224-241, 244-262, 281-323, 342-351, 359-367, 369-396, 406-416,424-433, 450-456, 485-491, 493-499, 501-515, 517-535 and 289-305 of SeqID No 212; 4-17, 22-44, 53-60, 66-83, 87-94, 101-106, 110-116, 131-137,148-183, 189-207, 209-215, 233-242, 251-262, 264-272, 290-296, 308-327,359-373, 375-380, 397-405, 415-420, 426-433, 444-475, 478-484, 529-536,548-558 and 106-126 of Seq ID No 213; 4-38, 42-50, 58-64, 72-81, 92-118,140-146, 157-165, 172-192, 198-204, 208-216, 227-234, 238-258, 271-278,288-293, 311-322, 327-346, 357-370, 375-383, 395-409, 411-417, 425-432,436-445, 109-129 and 370-380 of Seq ID No 214; 23-30, 36-49, 52-64,86-94, 97-104, 121-129, 257-272, 279-286, 288-294, 307-327, 334-340,369-375, 377-386, 406-412, 418-423, 430-438, 441-447, 459-465, 469-476,482-488, 510-546, 550-580, 584-622, 638-645, 653-659, 675-683, 692-705,723-731, 752-761, 788-795 and 54-72 of Seq ID No 215; 11-33, 36-46,88-104, 116-126, 134-170, 189-195, 199-217, 225-250, 255-261, 266-273,280-291, 296-313, 334-341, 343-349, 354-360, 362-369, 373-380, 387-401,406-420 and 259-273 of Seq ID No 216; 9-14, 28-44, 57-64, 72-79, 86-93,104-111, 116-126, 142-150, 159-164 and 61-86 of Seq ID No 217; 10-17,26-33, 43-61, 69-95, 101-107, 109-125, 129-135, 137-144, 147-153,158-169, 177-187, 209-219, 221-232, 235-247, 261-268, 271-282, 296-302,306-347, 355-362, 364-379, 386-399, 409-418, 424-442, 451-460, 467-479,490-498 and 60-74 of Seq ID No 218; 8-14, 20-31, 65-84, 94-99, 154-179,193-207, 238-253 and 96-118 of Seq ID No 219; 4-24, 30-44, 47-62, 84-93,108-116, 124-133, 136-141, 201-209, 217-223, 228-235, 238-245, 247-270,275-285, 290-314, 328-338, 342-349, 353-365, 375-383, 386-392, 394-402,417-427, 443-459, 465-481, 492-514, 516-524, 550-566, 602-617, 630-639,666-676, 687-693, 719-730, 747-753, 783-790, 799-816, 824-831, 837-842and 167-189 of Seq ID No 220; 6-15, 18-28, 58-66, 84-101, 106-129,136-151, 154-165, 182-203, 205-211, 214-220, 222-228, 233-240, 251-260,270-277, 284-291, 306-315, 322-328, 363-369, 378-388, 392-405, 443-452,495-501, 512-523, 574-583 and 362-375 of Seq ID No 221; 5-25, 27-34,47-59, 64-70, 76-86, 145-158, 166-183, 189-202, 217-231, 235-242,260-270, 278-309 and 1-102 of Seq ID No 222; 4-19, 24-76, 78-83, 90-99,102-109, 114-122, 137-147, 154-174, 177-188, 203-212, 217-223, 227-239and 226-325 of Seq ID No 223; 7-37, 71-90, 94-109, 117-128, 141-153,179-192, 199-206, 225-231, 237-243, 258-264 and 40-51 of Seq ID No 224;13-19, 25-30, 46-59, 75-91, 101-107, 114-124, 129-135, 137-145, 160-167,171-179, 187-194, 209-215, 217-222, 229-239, 243-249, 257-265, 269-275,299-308, 310-327 and 282-300 of Seq ID No 225; 86-100, 216-230, 342-369,382-388, 424-430, 438-445, 452-458, 488-494, 501-518, 554-560, 568-574,584-592, 603-609, 611-629, 639-645, 652-661, 669-699, 708-714, 726-738,747-753, 763-775, 785-791, 794-807, 815-824, 826-845, 854-860, 863-868,870-883, 892-898, 901-906, 909-921, 930-937, 946-959, 968-974, 977-990,998-1007, 1009-1027, 1037-1043, 1046-1051, 1053-1066, 1075-1081,1084-1089, 1092-1103, 1113-1119, 1122-1135, 1143-1152, 1154-1172,1182-1188, 1191-1196, 1200-1210, 1220-1226, 1229-1235, 1237-1249,1259-1265, 1268-1281, 1289-1298, 1305-1318, 1328-1334, 1337-1343,1345-1357, 1367-1373, 1390-1396, 1405-1411, 1418-1423, 1426-1435,1445-1455, 1474-1483, 1493-1500, 1505-1512, 1517-1524, 1538-1544,1568-1578, 1595-1601, 1674-1682, 1687-1720, 1728-1736, 1738-1744,1754-1761, 1764-1774, 1798-1824, 1836-1842, 1886-1893, 1895-1903,366-781, 782-1518 and 1731-1747 of Seq ID No 226; 4-17, 20-39, 46-55,60-66, 102-110, 114-122, 125-131, 161-167, 172-178, 185-190, 195-202,218-232, 236-252, 264-291, 293-302, 309-315, 324-339 and 169-381 of SeqID No 227; 5-10, 13-40, 42-53, 69-75, 83-89, 120-135, 150-161, 174-190,203-225, 229-247, 257-287, 318-348 and 30-200 of Seq ID No 228; 7-19,43-53, 64-72, 124-139, 52-84 and 120-131 of Seq ID No 229; 12-19, 39-48,58-100, 117-123, 154-162, 164-187, 189-195, 202-216, 218-235, 241-246,262-278, 315-328, 333-347, 354-366, 372-379, 391-405, 422-429, 431-442,444-450, 458-466, 478-485, 494-501, 504-510, 520-535, 573-580, 589-598,615-625, 666-676, 686-698, 722-729, 737-746, 756-767, 787-796, 805-816,824-829, 833-848, 856-864, 866-876, 879-886, 898-904, 918-924, 927-934,941-960, 967-978 and 561-575 of Seq ID No 230; 11-29, 49-55, 70-77,84-100, 102-112, 148-155, 160-177, 181-204 and 1-104 of Seq ID No 231;27-44, 64-71, 122-133, 151-156, 164-178, 214-220, 226-232, 235-244,253-262, 282-288, 294-310, 317-325, 350-356, 362-368, 376-383, 438-443,449-454, 459-464, 492-498, 500-511, 529-535, 538-546, 567-573, 597-603,660-665, 674-679, 724-734, 763-769, 773-784, 791-801, 807-815, 821-826,840-848, 863-868, 897-902, 908-928, 932-953, 956-975, 980-987, 990-996,1012-1018, 1042-1063, 1095-1116, 1149-1157, 1160-1167, 110-357, 358-501and 502-1161 of Seq ID No 232; 4-21, 64-71, 73-84, 128-138, 144-162,203-217, 240-263, 288-298, 300-308, 310-317, 325-351, 369-380, 391-411and 330-345 of Seq ID No 233; 5-11, 25-31, 39-48, 51-79, 89-98, 100-122,135-148, 166-201, 203-227, 230-250, 254-260, 266-272, 274-282, 299-305,328-337 and 31-45 of Seq ID No 234; 12-23, 29-48, 51-60, 66-72, 75-81,83-93, 103-115, 133-148, 168-174, 195-204, 222-229, 231-240, 242-251,270-280, 286-305, 322-344, 349-360, 364-370, 378-400, 421-441, 448-484,486-493, 495-501, 504-534, 547-561, 567-590, 597-607, 621-635, 643-649,658-685, 688-694, 702-711, 717-731, 737-742, 759-765, 767-772, 776-786,803-809, 815-825, 854-908, 910-919, 923-930, 942-948, 961-975, 994-1014and 915-940 of Seq ID No 235; 4-9, 32-47, 51-61, 75-96, 139-191 and1-124 of Seq ID No 236; 4-13, 17-38, 43-49, 55-76, 88-95, 110-121,128-146, 151-157, 162-214, 222-240, 243-249, 251-273, 275-281, 292-298,300-309, 312-320, 322-331, 355-369, 376-408, 446-460, 471-482, 485-509and 191-203 of Seq ID No 237; 4-21, 72-82, 89-103, 106-115, 118-124,140-146, 174-184, 191-200, 204-213, 218-224, 261-266, 282-293, 299-309,311-340, 342-358, 362-372, 381-389, 391-402, 413-421, 438-447, 457-464,470-478, 501-507, 545-560, 578-624, 631-641, 658-670, 680-689, 717-738,753-759, 795-805, 816-822, 830-838, 842-848, 869-881, 892-898, 33-51 and818-835 of Seq ID No 238; 4-21, 79-85, 156-177, 183-188, 206-214,243-249, 261-269, 287-292, 315-322, 334-345, 360-366, 374-390, 402-411,37-97 and 260-399 of Seq ID No 239; 4-9, 19-54, 58-78, 97-104, 111-120,126-134, 137-145, 163-173, 178-188, 193-203, 211-224, 246-286, 288-324,337-346, 355-362, 374-390, 392-398, 409-417 and 240-249 of Seq ID No240; 5-12, 14-31, 35-41, 43-61, 82-92, 97-105, 134-145, 155-166,184-203, 215-223, 225-251, 272-279, 281-306, 310-345, 358-418, 435-473,482-490, 525-532, 538-547, 549-563, 578-604, 613-639 and 144-154 of SeqID No 241; 53-59, 64-72, 74-100, 133-152, 154-172, 176-181, 207-214,225-238, 275-297, 304-310, 331-340, 362-367, 384-395, 403-410, 437-443,448-456, 482-490, 579-597, 602-610, 625-630, 633-651, 699-707, 709-715,734-743, 750-762 and 544-685 of Seq ID No 242; 12-18, 22-40, 45-83,89-97, 103-109, 147-153, 159-173, 195-204, 210-219, 243-253, 259-265,273-282, 303-309, 315-325, 332-340, 346-358, 362-367, 377-390, 393-402,418-426, 447-455, 467-480, 505-512, 514-525, 548-561, 566-576, 584-596,619-626, 638-645, 649-659, 661-680, 699-708, 714-720, 753-759, 766-772,775-781, 801-808, 202-218, 282-299, 339-350 and 617-628 of Seq ID No243; 5-33, 52-62, 87-101, 111-135, 137-143, 145-152, 190-202, 209-221,233-245, 253-270 and 151-215 of Seq ID No 244; 19-29, 32-39, 42-48,75-94, 124-135, 137-145, 152-160, 176-182, 193-203, 215-236, 266-273,275-291, 297-306, 311-319, 322-342, 348-360, 369-378, 394-401 and 48-64of Seq ID No 245; 4-11, 13-33, 36-43, 53-63, 65-80, 112-129, 134-141,143-155, 157-168, 178-188, 191-199, 201-207, 215-229, 242-255, 263-270,283-315, 320-329, 333-338, 340-349, 412-426, 465-478, 485-490, 498-512,540-554 and 390-516 of Seq ID No 246; 4-18, 23-32, 41-47, 54-70, 88-99,104-111, 118-138, 143-148, 150-162, 168-175, 181-188, 203-211, 214-220,227-245, 251-268, 275-281, 287-296, 323-333 and 1-90 of Seq ID No 247;8-34, 38-49, 72-83, 85-91, 94-104, 112-125, 134-142, 148-168, 181-189,191-198, 202-214, 222-233, 242-254, 256-262, 273-278, 287-294, 314-325and 141-159 of Seq ID No 248; 4-24, 30-36, 47-75, 82-105, 124-134,151-157, 192-202, 208-214, 219-226, 234-247, 285-290, 318-324, 332-340,343-349, 380-386, 453-462, 472-478, 484-501, 531-540, 550-557, 604-612,620-625, 642-648, 652-671, 64-84, 93-180 and 181-446 of Seq ID No 249;12-18, 24-32, 68-75, 77-83, 96-101, 109-116, 129-136, 152-164, 175-184,190-199, 206-215, 224-233, 241-250, 258-264, 273-292, 302-312, 319-331,334-346, 348-368, 387-395, 408-416, 420-429, 437-452 and 364-374 of SeqID No 250; 11-28, 36-52, 60-67, 74-79, 108-116 and 61-76 of Seq ID No251; 20-27, 38-49, 69-74, 84-107, 138-145, 161-168, 179-195, 210-226,228-252, 267-281, 283-296, 305-311, 333-340, 342-356, 361-372, 380-399,401-414, 458-466, 475-481, 492-507, 515-520 and 146-160 of Seq ID No252; 43-61, 68-74, 76-90, 120-128, 130-149, 156-161, 164-182, 206-234,242-252, 269-274, 291-304, 332-345, 349-355, 360-371, 374-388, 434-440,447-453, 459-465, 469-496, 504-522 and 261-285 of Seq ID No 253; 4-17,24-30, 37-49, 87-98, 118-124, 126-136, 144-171, 176-188, 206-214,216-228, 233-240, 246-252, 262-271, 277-297, 307-330, 333-342, 346-352,355-361, 368-386, 391-400, 413-420, 474-480 and 401-427 of Seq ID No254; 15-26, 31-46, 51-72, 80-93, 96-109, 131-137, 150-158, 179-185,189-209, 211-219, 221-234, 241-247, 255-262, 265-271, 283-288 and173-190 of Seq ID No 255; 28-37, 39-45, 51-58, 77-84, 89-97, 132-148,171-180, 199-205, 212-218, 220-226, 257-265, 273-300, 307-327, 334-340,344-365, 385-390, 402-408, 426-436, 450-468, 476-485 and 425-497 of SeqID No 256; 4-25, 70-76, 80-88, 90-100, 120-128, 162-169, 183-203,261-277, 279-289, 291-297, 302-308, 321-327, 339-353, 358-377, 392-401,404-410, 414-422, 443-450, 456-461, 470-488, 490-497, 510-535, 570-611,618-630, 639-647, 649-660, 668-690, 702-716, 718-724, 737-747, 750-764and 497-509 of Seq ID No 257; 12-48, 50-64, 99-108, 216-223, 235-241,244-254, 262-274, 287-293, 310-316, 320-326, 361-366, 377-383, 390-395,408-414, 418-425, 438-444, 462-469, 494-505, 524-530, 536-547, 551-566,592-598, 601-613, 678-685, 687-695, 709-717, 727-737, 751-757, 760-765,772-778, 782-788, 801-807, 822-830, 859-868, 870-878, 884-890, 898-903,909-919, 953-969, 973-980, 990-1000, 1002-1019, 1041-1047, 1059-1065,1090-1095, 1116-1127, 1130-1139, 1143-1149, 1151-1168, 1178-1183,1188-1195, 1197-1209, 1213-1220, 1226-1234, 1236-1247, 1255-1274,1276-1282, 76-100, 270-284, 309-438, 493-505, 786-942 and 947-967 of SeqID No 258; 4-9, 24-34, 46-95, 97-109, 119-130 and 138-156 of Seq ID No259; 9-26, 28-35, 43-53, 55-68, 83-92, 99-105, 110-135, 139-149,157-162, 164-170, 173-183, 193-208, 210-230, 239-245, 253-259, 263-271,293-305, 310-320, 322-331, 336-343, 351-364, 367-376, 92-107 and 154-173of Seq ID No 260; 19-39, 52-62, 108-117, 145-152, 160-168, 194-203,229-240, 252-268, 280-287, 308-316, 333-339, 383-390, 403-412, 414-424,438-445, 464-472, 479-484, 489-505, 510-526 and 247-260 of Seq ID No261; 5-17, 25-52, 60-77, 105-113, 118-125, 162-167, 228-234, 272-279,328-334, 341-357, 381-395, 400-406, 512-518, 557-569, 586-592, 645-651,690-695, 701-709, 720-726, 733-743, 751-758, 781-786, 879-886, 929-934,939-944, 952-960, 965-975, 994-1001, 1039-1045, 1102-1109, 1164-1181,1198-1206, 1223-1229, 1253-1259, 1283-1292, 1312-1317, 1339-1349,1360-1370, 1389-1398, 1400-1412, 1452-1465, 1470-1484, 1490-1497,1519-1525, 1554-1564, 1578-1591, 1623-1636, 1638-1646, 1669-1679,1685-1697, 1704-1711, 1713-1720, 1730-1736, 1738-1749, 1756-1764,1778-1786, 1796-1803, 1817-1826, 1849-1866, 1975-1993, 2017-2032,2044-2053, 2070-2086, 2091-2109, 2116-2127, 2156-2167, 2182-2188,2197-2202, 2244-2252, 2281-2287, 2290-2307, 2350-2361, 2383-2404,2425-2433, 2445-2455, 2495-2505 and 394-549 of Seq ID No 262; 9-24,31-53, 57-67, 69-79, 84-114, 133-141, 144-172, 178-186 and 13-46 of SeqID No 263; 4-25, 27-35, 43-52, 59-70, 79-91, 115-130, 136-152, 154-163,170-179 and 1-58 of Seq ID No 264; 4-30, 49-55, 71-80, 96-105, 111-126,139-146, 149-162, 239-245, 279-285, 290-296, 300-307, 331-337, 343-350and 250-351 of Seq ID No 265; 9-27, 34-41, 43-51, 92-111, 114-120,123-131, 139-150, 156-171, 176-186, 188-204, 229-241, 252-258, 266-279,288-297, 319-334, 338-348, 373-379, 389-398, 431-439, 479-484 and214-398 of Seq ID No 266; 4-15, 18-27, 47-52, 68-83, 91-97, 104-110,115-121, 139-147, 157-164, 198-206, 227-236, 241-254, 264-273, 278-289,311-320, 353-361, 372-383, 405-420, 426-434 and 232-386 of Seq ID No267; 4-10, 24-34, 91-97, 129-141, 156-163, 184-190, 205-219, 229-235,256-273, 278-285 and 93-116 of Seq ID No 268; 7-29, 35-54, 71-83, 85-91,104-111, 122-134, 138-144, 146-154, 158-174, 177-183, 186-201, 207-215,223-235, 240-247, 262-273, 275-283, 287-292 and 48-66 of Seq ID No 269;7-27, 31-47, 49-70, 75-102, 110-149, 157-171, 217-223, 235-251, 294-302,358-364, 367-375, 387-393, 395-412, 423-430, 441-451, 456-470, 472-486,488-495, 499-509, 515-529, 536-549, 556-570, 574-603, 607-615, 625-633,642-658, 670-676, 683-702, 708-716, 720-726, 747-756, 763-784, 803-812,815-826 and 475-490 of Seq ID No 270; 7-22, 30-38, 53-59, 64-75, 83-95,97-112, 120-131, 133-142, 145-151, 154-166, 172-180, 189-203, 227-238,277-287, 9-156 and 174-287 of Seq ID No 271; 13-23, 25-32, 111-117,150-164, 185-193, 207-212, 216-224, 230-236, 263-272, 304-311, 342-348,374-385, 391-407, 444-458, 480-487, 489-499, 523-542, 544-558, 572-579,620-640, 686-696, 703-710, 742-755, 765-772, 817-822, 830-837, 865-872,931-937 and 66-86 of Seq ID No 272; 4-27, 49-56, 62-70, 86-92, 121-127,151-163, 170-182, 195-202, 212-226, 237-243 and 234-254 of Seq ID No273; 4-10, 13-24, 39-51, 62-78, 92-104, 107-117, 134-141, 156-161,166-181, 210-216, 222-229, 256-266, 273-280, 297-304, 313-330, 336-349,371-376, 433-439, 443-448, 488-493, 506-515, 527-534, 560-572, 575-583,587-593 and 252-483 of Seq ID No 274; 4-15, 21-38, 45-56, 81-95,102-108, 118-130, 133-147, 152-162, 166-171, 199-204, 211-218, 230-240,253-261, 274-283, 288-294, 312-317, 325-336, 344-357, 391-414 and 24-146of Seq ID No 275; 26-31, 38-56, 65-82, 90-101, 112-119, 123-153,175-188, 197-216, 234-242, 249-265, 273-286, 290-305, 327-335, 338-346,361-372, 394-404 and 290-306 of Seq ID No 276; 17-26, 43-48, 50-73,81-93, 95-107, 139-146, 158-168, 171-176, 190-196, 202-212, 216-223,243-266, 274-282, 308-313, 324-330, 344-378, 380-387, 403-422, 427-443,448-455, 457-465, 491-515, 517-528, 553-567, 589-599, 610-617, 642-648,670-697, 709-717, 726-743, 745-759, 769-803, 807-823, 840-849 and820-851 of Seq ID No 277; 4-18, 39-48, 53-63, 66-90, 102-117, 125-134,137-145, 156-162, 169-197, 26-40 and 56-80 of Seq ID No 278; 21-33,36-42, 49-60, 68-76, 91-105, 123-130, 141-161, 169-178, 185-190,192-199, 205-214, 223-233, 239-247, 260-269, 284-293, 300-314, 324-352,357-364, 373-382, 389-403, 420-432, 438-446, 466-471, 477-484, 503-509,549-556, 558-576, 600-623, 625-635, 654-661, 663-669, 671-687, 702-716,735-741, 744-750, 757-766, 776-786, 807-815, 824-832, 854-860, 863-897,909-915, 920-946, 952-959, 982-997, 1024-1038, 1049-1055, 1071-1085,1104-1113, 1121-1132, 1138-1150, 1187-1196, 1212-1221, 1227-1236,1257-1262, 1264-1278, 1282-1294, 1307-1318, 1353-1370, 1382-1388,1396-1409, 1434-1440, 1446-1454, 1465-1478, 1485-1513, 1516-1529,1540-1545, 1563-1568, 1575-1593, 1607-1616, 1628-1645, 1648-1661,1676-1682, 1689-1697, 1713-1719, 1739-1749, 1753-1758, 1763-1774,1797-1803, 1807-1846, 1855-1874, 1877-1891, 1893-1907, 1912-1925,1931-1943, 1955-1965, 1976-1990, 2032-2043, 2045-2051, 2099-2105,2131-2138, 2161-2179, 2188-2199, 2205-2216, 2219-2227, 2235-2245,2247-2267, 2277-2288, 2294-2304, 2314-2326, 2346-2358, 2365-2377,2383-2402, 2407-2423, 2437-2450, 2454-2473, 2489-2497, 2525-2531,2557-2570, 2580-2587, 2589-2599, 2621-2641, 2647-2653, 2661-2677,2685-2690, 2697-2717, 2722-2733, 2739-2777, 2786-2793, 2801-2808,2811-2822, 2825-2835, 2838-2845, 2859-2871, 2877-2883, 213-344, 954-1080and 2524-2733 of Seq ID No 279; 10-16, 18-23, 28-41, 63-69, 77-91,101-109, 118-136, 146-153, 155-162, 168-179, 192-207, 217-226, 229-235,239-254, 279-286, 294-307, 313-319, 334-341, 344-353, 363-377, 390-396and 178-328 of Seq ID No 280; 18-42, 68-84, 89-95, 100-105, 107-115,125-135, 154-177, 189-195, 205-228, 236-243, 252-259, 279-300, 309-316,323-331, 340-351, 353-364, 377-402 and 85-97 of Seq ID No 281; 4-18,26-32, 66-76, 100-126, 151-159, 178-186, 188-194, 200-210, 241-248,253-259, 262-279, 284-291, 307-313, 315-322, 327-337, 376-386, 399-407,432-441, 467-473, 487-497, 499-505, 543-549, 560-568, 585-593, 598-604,608-614, 630-642, 647-653, 690-703, 717-730, 21-200 and 468-480 of SeqID No 282; 17-49, 52-58, 62-73, 78-97, 100-117, 122-172, 185-190,193-217, 225-236 and 33-42 of Seq ID No 283; 7-39, 50-58, 73-89, 96-107,109-120, 126-142, 152-170, 178-202, 205-211, 224-244, 249-259, 261-270,300-310, 312-325 and 158-169 of Seq ID No 284; 4-31, 40-64, 71-82,85-92, 102-124, 126-139, 147-152, 159-173, 176-188, 195-207, 210-216,234-241, 249-256, 258-276, 279-293, 296-302, 310-315, 349-356, 363-378,380-403, 411-426, 435-441, 448-459, 463-476, 488-494 and 201-221 of SeqID No 285; 5-13, 15-74, 87-104, 107-120, 123-129, 136-145, 150-191,193-206, 227-248, 250-264, 278-302, 304-323, 332-378, 384-407, 409-419,425-457, 462-471, 474-497, 511-545, 555-564, 571-578, 585-598, 640-647,669-675, 682-691, 693-705, 729-743, 752-761, 772-780, 786-804, 808-818,822-846, 858-880, 884-900, 910-939, 941-947, 962-971, 973-988, 998-1003,1007-1027 and 236-259 of Seq ID No 286; 4-19, 27-8, 81-111, 121-160 and60-79 of Seq ID No 287; 4-37, 40-46, 52-57, 199-205, 222-229, 236-244,250-267, 269-282 and 27-197 of Seq ID No 288; 4-16, 24-30, 32-38, 63-75,86-92, 98-111, 113-126, 160-165, 170-180, 198-204, 227-233, 239-245,253-273, 308-314, 352-365, 382-387, 395-403, 423-429, 472-482, 484-493,501-507, 518-526, 536-541, 543-550, 556-562, 586-600, 626-633, 649-661,680-688 and 546-559 of Seq ID No 289; 16-33, 48-59, 63-71, 77-92,94-109, 117-124, 139-151, 169-181, 184-227, 233-249, 251-261, 263-275,282-294, 297-321, 326-332, 341-355, 383-399 and 258-272 of Seq ID No290; 11-26, 31-39, 43-52, 55-62, 64-70, 80-94, 123-133, 135-141,172-181, 185-206, 209-218, 224-230, 238-244, 251-262, 264-271, 290-301,306-324, 333-340, 350-357, 367-375, 390-397, 434-441, 443-448, 77-226and 350-429 of Seq ID No 291; 4-13, 22-27, 31-45, 50-59, 72-96, 99-114,131-141, 143-150, 159-176, 180-186, 189-198, 208-214, 234-253, 271-287,294-299, 310-366, 382-390, 398-416, 424-443 and 283-305 of Seq ID No292; 9-26, 30-53, 62-72, 86-95, 112-122, 136-145, 153-160, 209-221,227-237, 241-268, 281-288, 291-298, 308-314, 321-328, 336-346, 351-379,388-397, 409-416, 423-433, 443-481, 511-519 and 213-232 of Seq ID No293; 12-18, 25-31, 38-50, 59-67, 71-82, 96-126 and 76-88 of Seq ID No294; 4-25, 39-44, 64-71, 74-88, 100-113, 128-138, 151-162, 164-177,185-190, 204-213, 233-239, 246-254, 281-286, 293-306, 309-318, 333-347,349-359, 385-398, 404-423, 458-465, 477-484, 490-499, 501-533, 554-566,582-590, 596-616, 624-629, 631-639, 654-680, 694-720, 735-743 and 2-100of Seq ID No 295; 4-16, 36-41, 52-75, 98-107, 109-117, 122-128, 133-139,141-155, 159-165, 169-182, 187-193, 195-201, 211-224, 230-236, 247-269,278-290 and 75-92 of Seq ID No 296; 7-21, 25-33, 37-43, 87-94, 103-120,131-147, 168-174, 197-203, 207-212, 227-237, 247-257, 263-271, 279-287,298-306, 320-325, 332-340, 363-374, 379-384, 390-401, 403-414, 428-433,448-457, 462-475, 483-390, 513-519, 525-535, 543-554, 559-566, 571-620,625-631, 636-642, 659-670, 688-706, 708-723, 770-779, 787-793, 796-807,820-840, 848-854, 863-874, 895-905, 912-919, 934-942, 968-975, 983-1000,1012-1019, 1026-1036, 1050-1060, 1064-1070, 1081-1091, 1094-1108,1112-1118, 1140-1152, 1164-1169, 1172-1180, 1187-1192 and 732-748 of SeqID No 297; 23-40, 42-59, 66-73, 78-97, 111-128, 130-141, 157-166,178-183 and 53-71 of Seq ID No 298; 4-27, 38-44, 47-57, 59-85, 99-106,114-121, 154-166, 181-186, 193-198, 238-244, 253-262, 272-278, 287-299,314-320, 338-350, 358-368, 382-388, 407-416, 433-446, 456-461, 463-473and 86-195 of Seq ID No 299; 5-24, 38-59, 64-80, 87-99, 105-126,134-142, 149-163, 165-179, 181-202, 205-220, 227-233, 243-250, 257-263and 87-245 of Seq ID No 300; 5-32, 47-53, 66-79, 81-97, 115-151,155-174, 183-188, 196-210, 215-226, 230-238, 253-258, 263-270, 276-282,295-301, 304-325, 334-344, 360-390, 397-412, 425-432, 434-462, 478-494,508-526, 539-564, 571-579, 347-371 and 375-386 of Seq ID No 301; 4-15,36-44, 49-56, 60-66, 68-82, 84-103, 109-115, 118-141, 147-154, 160-168,176-185 and 26-39 of Seq ID No 302; 7-13, 23-33 and 13-21 of Seq ID No303; 2-10 of Seq ID No 304; 4-9, 12-18, 35-42, 49-62 and 6-18 of Seq IDNo 305; 19-25 and 1-13 of Seq ID No 306; 15-21, 27-45 and 12-25 of SeqID No 307; 14-20 and 1-14 of Seq ID No 308; 4-18 and 13-26 of Seq ID No309; 8-21 and 2-20 of Seq ID No 310; 4-14 and 4-16 of Seq ID No 311;3-12 of Seq ID No 312; 6-14, 6-25, 35-57 and 2-14 of Seq ID No 313;6-25, 35-57 and 17-31 of Seq ID No 314; 14-25, 32-46 and 5-19 of Seq IDNo 315; 18-31 and 5-16 of Seq ID No 316; 19-24 and 4-26 of Seq ID No317; 13-21, 29-34, 47-58, 61-73 and 36-47 of Seq ID No 318; 4-15 and5-24 of Seq ID No 319; 6-18 of Seq ID No 320; 13-20 and 4-13 of Seq IDNo 321; 15-23 of Seq ID No 322; 4-9 and 7-21 of Seq ID No 323; 1-10 ofSeq ID No 324; 4-14 of Seq ID No 325; 4-17, 35-41, 46-89, 93-98 and70-88 of Seq ID No 326; 1-13 of Seq ID No 327; 4-16, 26-32 and 25-38 ofSeq ID No 328; 8-15, 23-28 and 4-17 of Seq ID No 329; 4-12 and 1-15 ofSeq ID No 330; 4-29, 31-42, 52-58 and 6-16 of Seq ID No 331; 4-9, 24-32and 9-19 of Seq ID No 332; 4-12, 18-27 and 5-18 of Seq ID No 333; 4-11,37-56, 58-92 and 18-29 of Seq ID No 334; 8-28 and 20-35 of Seq ID No335; 4-15 of Seq ID No 336; 4-23, 27-39, 55-63 and 35-58 of Seq ID No337; 6-26, 28-54 and 28-47 of Seq ID No 338; 4-10, 38-52, 58-82 and30-49 of Seq ID No 339; 4-22, 29-35, 44-50, 53-68, 70-80 and 20-33 ofSeq ID No 340; 22-28, 30-36 and 18-33 of Seq ID No 341; 4-11, 13-21,25-30 and 20-30 of Seq ID No 342; 10-22 and 10-23 of Seq ID No 343; 4-11and 9-20 of Seq ID No 344; 14-25, 32-46 and 6-19 of Seq ID No 345; 5-30and 14-33 of Seq ID No 346; 4-15, 28-35, 46-55, 59-65, 76-84 and 9-24 ofSeq ID No 347; 27-33 and 5-19 of Seq ID No 348; 5-13 and 8-18 of Seq IDNo 349; 9-22, 24-34 and 21-40 of Seq ID No 350; 4-17, 35-41, 46-89,93-98 and 71-89 of Seq ID No 351; 4-12, 14-24 and 2-17 of Seq ID No 352;9-17 and 5-16 of Seq ID No 353; 7-41, 48-58, 63-75, 80-89 and 43-53 ofSeq ID No 354; 4-22, 25-30 and 4-14 of Seq ID No 355; 4-55 and 18-33 ofSeq ID No 356; 262-280 of Seq ID No 179; 131-146 of Seq ID No 186;207-224 of Seq ID No 188; 27-50, 203-217 and 313-325 of Seq ID No 189;110-129 of Seq ID No 192; 156-179, 174-197, 192-215, 210-233, 228-251and 246-267 of Seq ID No 190; 377-400 of Seq ID No 196; 34-43, 234-257and 350-367 of Seq ID No 203; 304-327 of Seq ID No 207; 25-48, 43-66 and61-82 of Seq ID No 222; 398-421, 416-439, 434-457, 452-475, 470-493,488-511, 506-529, 524-547, 621-644, 639-664, 707-730, 725-748, 743-766,761-784, 779-802, 797-820, 984-1007, 1002-1025, 1020-1043, 1038-1061,1056-1079, 1074-1097, 1092-1115, 1286-1309, 1304-1327, 1322-1345,1340-1363, 1358-1381, 1376-1399, 1394-1417, 1412-1435, 1430-1453,1448-1471, 1466-1489 and 1484-1507 of Seq ID No 226; 188-211, 206-229,224-247, 242-265, 260-283 and 278-296 of Seq ID No 227; 56-79 and122-132 of Seq ID No 229; 35-46 of Seq ID No 231; 178-201, 196-219,214-237, 232-255, 250-273, 268-291, 379-402, 397-420, 415-438, 433-456,451-474, 642-665, 660-683, 678-701, 696-719, 714-737, 732-755, 750-773,768-791, 899-922, 917-940, 935-958, 1037-1060, 1055-1078, 1073-1096 and1091-1114 of Seq ID No 232; 330-346 of Seq ID No 233; 571-594, 589-612,607-630, 625-648, 643-666 and 661-684 of Seq ID No 242; 188-207 of SeqID No 244; 61-84, 308-331, 326-349, 344-367, 362-385, 380-403 and398-421 of Seq ID No 249; 79-98, 345-366, 844-867, 870-887 and 890-905of Seq ID No 258; 94-109 of Seq ID No 268; 188-207 of Seq ID No 272;290-306 of Seq ID No 276; 826-849 of Seq ID No 277; 228-252, 247-270,265-288, 283-306, 301-324, 955-978, 973-996, 991-1014, 1009-1032,1027-1050, 1045-1068, 2533-2556, 2551-2574, 2569-2592, 2587-2610,2605-2628 and 2623-2646 of Seq ID No 279; 86-109 and 104-127 of Seq IDNo 288; 546-560 of Seq ID No 289; 260-271 of Seq ID No 290; 106-129,124-147, 142-165, 160-183, 178-201 and 375-398 of Seq ID No 291; 284-307of Seq ID No 292; 362-385 of Seq ID No 301, and fragments comprising atleast 6, preferably more than 8, especially more than 10 aa of saidsequences. All these fragments individually and each independently forma preferred selected aspect of the present invention.

All linear hyperimmune serum reactive fragments of a particular antigenmay be identified by analysing the entire sequence of the proteinantigen by a set of peptides overlapping by 1 amino acid with a lengthof at least 10 amino acids. Subsequently, non-linear epitopes can beidentified by analysis of the protein antigen with hyperimmune serausing the expressed full-length protein or domain polypeptides thereof.Assuming that a distinct domain of a protein is sufficient to form the3D structure independent from the native protein, the analysis of therespective recombinant or synthetically produced domain polypeptide withhyperimmune serum would allow the identification of conformationalepitopes within the individual domains of multi-domain proteins. Forthose antigens where a domain possesses linear as well as conformationalepitopes, competition experiments with peptides corresponding to thelinear epitopes may be used to confirm the presence of conformationalepitopes.

It will be appreciated that the invention also relates to, among others,nucleic acid molecules encoding the aforementioned fragments, nucleicacid molecules that hybridise to nucleic acid molecules encoding thefragments, particularly those that hybridise under stringent conditions,and nucleic acid molecules, such as PCR primers, for amplifying nucleicacid molecules that encode the fragments. In these regards, preferrednucleic acid molecules are those that correspond to the preferredfragments, as discussed above.

The present invention also relates to vectors, which comprise a nucleicacid molecule or nucleic acid molecules of the present invention, hostcells which are genetically engineered with vectors of the invention andthe production of hyperimmune serum reactive antigens and fragmentsthereof by recombinant techniques.

A great variety of expression vectors can be used to express ahyperimmune serum reactive antigen or fragment thereof according to thepresent invention. Generally, any vector suitable to maintain, propagateor express nucleic acids to express a polypeptide in a host may be usedfor expression in this regard. In accordance with this aspect of theinvention the vector may be, for example, a plasmid vector, a single ordouble-stranded phage vector, a single or double-stranded RNA or DNAviral vector. Starting plasmids disclosed herein are either commerciallyavailable, publicly available, or can be constructed from availableplasmids by routine application of well-known, published procedures.Preferred among vectors, in certain respects, are those for expressionof nucleic acid molecules and hyperimmune serum reactive antigens orfragments thereof of the present invention. Nucleic acid constructs inhost cells can be used in a conventional manner to produce the geneproduct encoded by the recombinant sequence. Alternatively, thehyperimmune serum reactive antigens and fragments thereof of theinvention can be synthetically produced by conventional peptidesynthesizers. Mature proteins can be expressed in mammalian cells,yeast, bacteria, or other cells under the control of appropriatepromoters. Cell-free translation systems can also be employed to producesuch proteins using RNAs derived from the DNA construct of the presentinvention.

Host cells can be genetically engineered to incorporate nucleic acidmolecules and express nucleic acid molecules of the present invention.Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, E. coli, Streptomyces and Bacillussubtillis cells; fungal cells, such as yeast cells and Aspergilluscells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells;animal cells such as CHO, COS, Hela, C127, 3T; 3, BHK, 293 and Bowesmelanoma cells; and plant cells.

The invention also provides a process for producing a H. pylorihyperimmune serum reactive antigen and a fragment thereof comprisingexpressing from the host cell a hyperimmune serum reactive antigen orfragment thereof encoded by the nucleic acid molecules provided by thepresent invention. The invention further provides a process forproducing a cell, which expresses a H. pylori hyperimmune serum reactiveantigen or a fragment thereof comprising transforming or transfecting asuitable host cell with the vector according to the present inventionsuch that the transformed or transfected cell expresses the polypeptideencoded by the nucleic acid contained in the vector.

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals but also additionalheterologous functional regions. Thus, for instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N- or C-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification or during subsequenthandling and storage. Also, regions may be added to the polypeptide tofacilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stability orto facilitate, purification, among others, are familiar and routinetechniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to solubilize orpurify polypeptides. For example, EP-A-O 464 533 (Canadian counterpart2045869) discloses fusion proteins comprising various portions ofconstant region of immunoglobin molecules together with another proteinor part thereof. In drug discovery, for example, proteins have beenfused with antibody Fc portions for the purpose of high-throughoutscreening assays to identify antagonists. See for example, {Bennett, D.et al., 1995} and {Johanson, K et al., 1995}.

The H. pylori hyperimmune serum reactive antigen or a fragment thereofcan be recovered and purified from recombinant cell cultures bywell-known methods including ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,hydroxylapatite chromatography and lectin chromatography.

The hyperimmune serum reactive antigens and fragments thereof accordingto the present invention can be produced by chemical synthesis as wellas by biotechnological means. The latter comprise the transfection ortransformation of a host cell with a vector containing a nucleic acidaccording to the present invention and the cultivation of thetransfected or transformed host cell under conditions, which are knownto the ones skilled in the art. The production method may also comprisea purification step in order to purify or isolate the polypeptide to bemanufactured. In a preferred embodiment the vector is a vector accordingto the present invention.

The hyperimmune serum reactive antigens and fragments thereof accordingto the present invention may be used for the detection of the organismor organisms in a sample containing these organisms or polypeptidesderived thereof. Preferably such detection is for diagnosis, morepreferable for the diagnosis of a disease, most preferably for thediagnosis of diseases related or linked to the presence or abundance ofGram-negative bacteria, especially bacteria selected from the groupcomprising Helicobacter, Campylobacter and Arcobacter. More preferably,the microorganisms are selected from the group comprising Helicobactercinaedi and Helicobacter fanneliae, especially the microorganism isHelicobacter pylori.

The present invention also relates to diagnostic assays such asquantitative and diagnostic assays for detecting levels of thehyperimmune serum reactive antigens and fragments thereof of the presentinvention in cells and tissues, including determination of normal andabnormal levels. Thus, for instance, a diagnostic assay in accordancewith the invention for detecting over-expression of the polypeptidecompared to normal control tissue samples may be used to detect thepresence of an infection, for example, and to identify the infectingorganism. Assay techniques that can be used to determine levels of apolypeptide, in a sample derived from a host are well-known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.Among these, ELISAs frequently are preferred. An ELISA assay initiallycomprises preparing an antibody specific to the polypeptide, preferablya monoclonal antibody. In addition, a reporter antibody generally isprepared which binds to the monoclonal antibody. The reporter antibodyis attached to a detectable reagent such as radioactive, fluorescent orenzymatic reagent, such as horseradish peroxidase enzyme.

The hyperimmune serum reactive antigens and fragments thereof accordingto the present invention may also be used for the purpose of or inconnection with an array. More particularly, at least one of thehyperimmune serum reactive antigens and fragments thereof according tothe present invention may be immobilized on a support. Said supporttypically comprises a variety of hyperimmune serum reactive antigens andfragments thereof whereby the variety may be created by using one orseveral of the hyperimmune serum reactive antigens and fragments thereofaccording to the present invention and/or hyperimmune serum reactiveantigens and fragments thereof being different. The characterizingfeature of such array as well as of any array in general is the factthat at a distinct or predefined region or position on said support or asurface thereof, a distinct polypeptide is immobilized. Because of thisany activity at a distinct position or region of an array can becorrelated with a specific polypeptide. The number of differenthyperimmune serum reactive antigens and fragments thereof immobilized ona support may range from as little as 10 to several 1000 differenthyperimmune serum reactive antigens and fragments thereof. The densityof hyperimmune serum reactive antigens and fragments thereof per cm² isin a preferred embodiment as little as 10 peptides/polypeptides per cm²to at least 400 different peptides/polypeptides per cm² and moreparticularly at least 1000 different hyperimmune serum reactive antigensand fragments thereof per cm².

The manufacture of such arrays is known to the one skilled in the artand, for example, described in U.S. Pat. No. 5,744,309. The arraypreferably comprises a planar, porous or non-porous solid support havingat least a first surface. The hyperimmune serum reactive antigens andfragments thereof as disclosed herein, are immobilized on said surface.Preferred support materials are, among others, glass or cellulose. It isalso within the present invention that the array is used for any of thediagnostic applications described herein. Apart from the hyperimmuneserum reactive antigens and fragments thereof according to the presentinvention also the nucleic acid molecules according to the presentinvention may be used for the generation of an array as described above.This applies as well to an array made of antibodies, preferablymonoclonal antibodies as, among others, described herein.

In a further aspect the present invention relates to an antibodydirected to any of the hyperimmune serum reactive antigens and fragmentsthereof, derivatives or fragments thereof according to the presentinvention. The present invention includes, for example, monoclonal andpolyclonal antibodies, chimeric, single chain, and humanized antibodies,as well as Fab fragments, or the product of a Fab expression library. Itis within the present invention that the antibody may be chimeric, i.e.that different parts thereof stem from different species or at least therespective sequences are taken from different species.

Antibodies generated against the hyperimmune serum reactive antigens andfragments thereof corresponding to a sequence of the present inventioncan be obtained by direct injection of the hyperimmune serum reactiveantigens and fragments thereof into an animal or by administering thehyperimmune serum reactive antigens and fragments thereof to an animal,preferably a non-human. The antibody so obtained will then bind thehyperimmune serum reactive antigens and fragments thereof itself. Inthis manner, even a sequence encoding only a fragment of a hyperimmuneserum reactive antigen and fragments thereof can be used to generateantibodies binding the whole native hyperimmune serum reactive antigenand fragments thereof. Such antibodies can then be used to isolate thehyperimmune serum reactive antigens and fragments thereof from tissueexpressing those hyperimmune serum reactive antigens and fragmentsthereof.

For preparation of monoclonal antibodies, any technique known in theart, which provides antibodies produced by continuous cell line culturescan be used. (as described originally in {Kohler, G. et al., 1975}.

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic hyperimmune serum reactive antigens and fragments thereofaccording to this invention. Also, transgenic mice, or other organismssuch as other mammals, may be used to express humanized antibodies toimmunogenic hyperimmune serum reactive antigens and fragments thereofaccording to this invention.

Alternatively, phage display technology or ribosomal display could beutilized to select antibody genes with binding activities towards thehyperimmune serum reactive antigens and fragments thereof either fromrepertoires of PCR amplified v-genes of lymphocytes from humans screenedfor possessing respective target antigens or from naïve libraries{McCafferty, J. et al., 1990}; {Marks, J. et al., 1992}. The affinity ofthese antibodies can also be improved by chain shuffling {Clackson, T.et al., 1991}.

If two antigen binding domains are present, each domain may be directedagainst a different epitope—termed ‘bispecific’ antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the hyperimmune serum reactive antigens and fragmentsthereof or purify the hyperimmune serum reactive antigens and fragmentsthereof of the present invention by attachment of the antibody to asolid support for isolation and/or purification by affinitychromatography.

Thus, among others, antibodies against the hyperimmune serum reactiveantigens and fragments thereof of the present invention may be employedto inhibit and/or treat infections, particularly bacterial infectionsand especially infections arising from H. pylori.

Hyperimmune serum reactive antigens and fragments thereof includeantigenically, epitopically or immunologically equivalent derivatives,which form a particular aspect of this invention. The term“antigenically equivalent derivative” as used herein encompasses ahyperimmune serum reactive antigen and fragments thereof or itsequivalent which will be specifically recognized by certain antibodieswhich, when raised to the protein or hyperimmune serum reactive antigenand fragments thereof according to the present invention, interfere withthe interaction between pathogen and mammalian host. The term“immunologically equivalent derivative” as used herein encompasses apeptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe interaction between pathogen and mammalian host.

The hyperimmune serum reactive antigens and fragments thereof, such asan antigenically or immunologically equivalent derivative or a fusionprotein thereof can be used as an antigen to immunize a mouse or otheranimal such as a rat or chicken. The fusion protein may providestability to the hyperimmune serum reactive antigens and fragmentsthereof. The antigen may be associated, for example by conjugation, withan immunogenic carrier protein, for example bovine serum albumin (BSA)or keyhole limpet haemocyanin (KLH). Alternatively, an antigenic peptidecomprising multiple copies of the protein or hyperimmune serum reactiveantigen and fragments thereof, or an antigenically or immunologicallyequivalent hyperimmune serum reactive antigen and fragments thereof, maybe sufficiently antigenic to improve immunogenicity so as to obviate theuse of a carrier.

Preferably the antibody or derivative thereof is modified to make itless immunogenic in the individual. For example, if the individual ishuman the antibody may most preferably be “humanized”, wherein thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in {Jones, P. et al., 1986} or {Tempest, P. et al., 1991}.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscle, delivery of DNA complexed withspecific protein carriers, coprecipitation of DNA with calciumphosphate, encapsulation of DNA in various forms of liposomes, particlebombardment {Tang D. et al., 1992}, {Eisenbraun, M. et al., 1993} and invivo infection using cloned retroviral vectors {Seeger, C. et al.,1984}.

In a further aspect the present invention relates to a peptide bindingto any of the hyperimmune serum reactive antigens and fragments thereofaccording to the present invention, and a method for the manufacture ofsuch peptides whereby the method is characterized by the use of thehyperimmune serum reactive antigens and fragments thereof according tothe present invention and the basic steps are known to the one skilledin the art.

Such peptides may be generated by using methods according to the stateof the art such as phage display or ribosome display. In case of phagedisplay, basically a library of peptides is generated, in form ofphages, and this kind of library is contacted with the target molecule,in the present case a hyperimmune serum reactive antigen and fragmentsthereof according to the present invention. Those peptides binding tothe target molecule are subsequently removed, preferably as a complexwith the target molecule, from the respective reaction. It is known tothe one skilled in the art that the binding characteristics, at least toa certain extent, depend on the particularly realized experimentalset-up such as the salt concentration and the like. After separatingthose peptides binding to the target molecule with a higher affinity ora bigger force, from the non-binding members of the library, andoptionally also after removal of the target molecule from the complex oftarget molecule and peptide, the respective peptide(s) may subsequentlybe characterised. Prior to the characterisation optionally anamplification step is realized such as, e.g. by propagating the peptideencoding phages. The characterisation preferably comprises thesequencing of the target binding peptides. Basically, the peptides arenot limited in their lengths, however, preferably peptides having alengths from about 8 to 20 amino acids are preferably obtained in therespective methods. The size of the libraries may be about 10² to 10¹⁸,preferably 10⁸ to 10¹⁵ different peptides, however, is not limitedthereto.

A particular form of target binding hyperimmune serum reactive antigensand fragments thereof are the so-called “anticalines” which are, amongothers, described in the German patent application DE 197 42 706.

In a further aspect the present invention relates to functional nucleicacids interacting with any of the hyperimmune serum reactive antigensand fragments thereof according to the present invention, and a methodfor the manufacture of such functional nucleic acids whereby the methodis characterized by the use of the hyperimmune serum reactive antigensand fragments thereof according to the present invention and the basicsteps are known to the one skilled in the art. The functional nucleicacids are preferably aptamers and spiegelmers.

Aptamers are D-nucleic acids, which are either single stranded or doublestranded and which specifically interact with a target molecule. Themanufacture or selection of aptamers is, e.g., described in Europeanpatent EP 0 533 838. Basically the following steps are realized. First,a mixture of nucleic acids, i.e. potential aptamers, is provided wherebyeach nucleic acid typically comprises a segment of several, preferablyat least eight subsequent randomised nucleotides. This mixture issubsequently contacted with the target molecule whereby the nucleicacid(s) bind to the target molecule, such as based on an increasedaffinity towards the target or with a bigger force thereto, compared tothe candidate mixture. The binding nucleic acid(s) are/is subsequentlyseparated from the remainder of the mixture. Optionally, the thusobtained nucleic acid(s) is amplified using, e.g. polymerase chainreaction. These steps may be repeated several times giving at the end amixture having an increased ratio of nucleic acids specifically bindingto the target from which the final binding nucleic acid is thenoptionally selected. These specifically binding nucleic acid(s) arereferred to as aptamers. It is obvious that at any stage of the methodfor the generation or identification of the aptamers samples of themixture of individual nucleic acids may be taken to determine thesequence thereof using standard techniques. It is within the presentinvention that the aptamers may be stabilized such as, e.g., byintroducing defined chemical groups which are known to the one skilledin the art of generating aptamers. Such modification may for examplereside in the introduction of an amino group at the 2′-position of thesugar moiety of the nucleotides. Aptamers are currently used astherapeutical agents. However, it is also within the present inventionthat the thus selected or generated aptamers may be used for targetvalidation and/or as lead substance for the development of medicaments,preferably of medicaments based on small molecules. This is actuallydone by a competition assay whereby the specific interaction between thetarget molecule and the aptamer is inhibited by a candidate drug wherebyupon replacement of the aptamer from the complex of target and aptamerit may be assumed that the respective drug candidate allows a specificinhibition of the interaction between target and aptamer, and if theinteraction is specific, said candidate drug will, at least inprinciple, be suitable to block the target and thus decrease itsbiological availability or activity in a respective system comprisingsuch target. The thus obtained small molecule may then be subject tofurther derivatisation and modification to optimise its physical,chemical, biological and/or medical characteristics such as toxicity,specificity, biodegradability and bioavailability.

Spiegelmers and their generation or manufacture is based on a similarprinciple. The manufacture of spiegelmers is described in internationalpatent application WO 98/08856. Spiegelmers are L-nucleic acids, whichmeans that they are composed of L-nucleotides rather than D-nucleotidesas aptamers are. Spiegelmers are characterized by the fact that theyhave a very high stability in biological systems and, comparable toaptamers, specifically interact with the target molecule against whichthey are directed. In the process of generating spiegelmers, aheterogeonous population of D-nucleic acids is created and thispopulation is contacted with the optical antipode of the targetmolecule, in the present case for example with the D-enantiomer of thenaturally occurring L-enantiomer of the hyperimmune serum reactiveantigens and fragments thereof according to the present invention.Subsequently, those D-nucleic acids are separated which do not interactwith the optical antipode of the target molecule. But those D-nucleicacids interacting with the optical antipode of the target molecule areseparated, optionally identified and/or sequenced and subsequently thecorresponding L-nucleic acids are synthesized based on the nucleic acidsequence information obtained from the D-nucleic acids. These L-nucleicacids which are identical in terms of sequence with the aforementionedD-nucleic acids interacting with the optical antipode of the targetmolecule, will specifically interact with the naturally occurring targetmolecule rather than with the optical antipode thereof. Similar to themethod for the generation of aptamers it is also possible to repeat thevarious steps several times and thus to enrich those nucleic acidsspecifically interacting with the optical antipode of the targetmolecule.

In a further aspect the present invention relates to functional nucleicacids interacting with any of the nucleic acid molecules according tothe present invention, and a method for the manufacture of suchfunctional nucleic acids whereby the method is characterized by the useof the nucleic acid molecules and their respective sequences accordingto the present invention and the basic steps are known to the oneskilled in the art. The functional nucleic acids are preferablyribozymes, antisense oligonucleotides and siRNA.

Ribozymes are catalytically active nucleic acids, which preferablyconsist of RNA which basically comprises two moieties. The first moietyshows a catalytic activity whereas the second moiety is responsible forthe specific interaction with the target nucleic acid, in the presentcase the nucleic acid coding for the hyperimmune serum reactive antigensand fragments thereof according to the present invention. Uponinteraction between the target nucleic acid and the second moiety of theribozyme, typically by hybridisation and Watson-Crick base pairing ofessentially complementary stretches of bases on the two hybridisingstrands, the catalytically active moiety may become active which meansthat it catalyses, either intramolecularly or intermolecularly, thetarget nucleic acid in case the catalytic activity of the ribozyme is aphosphodiesterase activity. Subsequently, there may be a furtherdegradation of the target nucleic acid, which in the end results in thedegradation of the target nucleic acid as well as the protein derivedfrom the said target nucleic acid. Ribozymes, their use and designprinciples are known to the one skilled in the art, and, for exampledescribed in {Doherty, E. et al., 2001} and {Lewin, A. et al., 2001}.

The activity and design of antisense oligonucleotides for themanufacture of a medicament and as a diagnostic agent, respectively, isbased on a similar mode of action. Basically, antisense oligonucleotideshybridise based on base complementarity, with a target RNA, preferablywith a mRNA, thereby activating RNase H. RNase H is activated by bothphosphodiester and phosphorothioate-coupled DNA. Phosphodiester-coupledDNA, however, is rapidly degraded by cellular nucleases with theexception of phosphorothioate-coupled DNA. These resistant,non-naturally occurring DNA derivatives do not inhibit RNase H uponhybridisation with RNA. In other words, antisense polynucleotides areonly effective as DNA RNA hybride complexes. Examples for this kind ofantisense oligonucleotides are described, among others, in US-patentU.S. Pat. No. 5,849,902 and U.S. Pat. No. 5,989,912. In other words,based on the nucleic acid sequence of the target molecule which in thepresent case are the nucleic acid molecules for the hyperimmune serumreactive antigens and fragments thereof according to the presentinvention, either from the target protein from which a respectivenucleic acid sequence may in principle be deduced, or by knowing thenucleic acid sequence as such, particularly the mRNA, suitable antisenseoligonucleotides may be designed base on the principle of basecomplementarity.

Particularly preferred are antisense-oligonucleotides, which have ashort stretch of phosphorothioate DNA (3 to 9 bases). A minimum of 3 DNAbases is required for activation of bacterial RNase H and a minimum of 5bases is required for mammalian RNase H activation. In these chimericoligonucleotides there is a central region that forms a substrate forRNase H that is flanked by hybridising “arms” comprised of modifiednucleotides that do not form substrates for RNase H. The hybridisingarms of the chimeric oligonucleotides may be modified such as by2′-O-methyl or 2′-fluoro. Alternative approaches used methylphosphonateor phosphoramidate linkages in said arms. Further embodiments of theantisense oligonucleotide useful in the practice of the presentinvention are P-methoxyoligonucleotides, partialP-methoxyoligodeoxyribonucleotides or P-methoxyoligonucleotides.

Of particular relevance and usefulness for the present invention arethose antisense oligonucleotides as more particularly described in theabove two mentioned US patents. These oligonucleotides contain nonaturally occurring 5′→3′-linked nucleotides. Rather theoligonucleotides have two types of nucleotides:2′-deoxyphosphorothioate, which activate RNase H, and 2′-modifiednucleotides, which do not. The linkages between the 2′-modifiednucleotides can be phosphodiesters, phosphorothioate orP-ethoxyphosphodiester. Activation of RNase H is accomplished by acontiguous RNase H-activating region, which contains between 3 and 52′-deoxyphosphorothioate nucleotides to activate bacterial RNase H andbetween 5 and 10 2′-deoxyphosphorothioate nucleotides to activateeucaryotic and, particularly, mammalian RNase H. Protection fromdegradation is accomplished by making the 5′ and 3′ terminal baseshighly nuclease resistant and, optionally, by placing a 3′ terminalblocking group.

More particularly, the antisense oligonucleotide comprises a 5′ terminusand a 3′ terminus; and from position 11 to 59 5′→3′-linked nucleotidesindependently selected from the group consisting of 2′-modifiedphosphodiester nucleotides and 2′-modified P-alkyloxyphosphotriesternucleotides; and wherein the 5′-terminal nucleoside is attached to anRNase H-activating region of between three and ten contiguousphosphorothioate-linked deoxyribonucleotides, and wherein the3′-terminus of said oligonucleotide is selected from the groupconsisting of an inverted deoxyribonucleotide, a contiguous stretch ofone to three phosphorothioate 2′-modified ribonucleotides, a biotingroup and a P-alkyloxyphosphotriester nucleotide.

Also an antisense oligonucleotide may be used wherein not the 5′terminal nucleoside is attached to an RNase H-activating region but the3′ terminal nucleoside as specified above. Also, the 5′ terminus isselected from the particular group rather than the 3′ terminus of saidoligonucleotide.

The nucleic acids as well as the hyperimmune serum reactive antigens andfragments thereof according to the present invention may be used as orfor the manufacture of pharmaceutical compositions, especially vaccines.Preferably such pharmaceutical composition, preferably vaccine is forthe prevention or treatment of diseases caused by, related to orassociated with H. pylori. In so far another aspect of the inventionrelates to a method for inducing an immunological response in anindividual, particularly a mammal, which comprises inoculating theindividual with the hyperimmune serum reactive antigens and fragmentsthereof of the invention, or a fragment or variant thereof, adequate toproduce antibodies to protect said individual from infection,particularly Helicobacter infection and most particularly H. pyloriinfections.

Yet another aspect of the invention relates to a method of inducing animmunological response in an individual which comprises, through genetherapy or otherwise, delivering a nucleic acid functionally encodinghyperimmune serum reactive antigens and fragments thereof, or a fragmentor a variant thereof, for expressing the hyperimmune serum reactiveantigens and fragments thereof, or a fragment or a variant thereof invivo in order to induce an immunological response to produce antibodiesor a cell mediated T cell response, either cytokine-producing T cells orcytotoxic T cells, to protect said individual from disease, whether thatdisease is already established within the individual or not. One way ofadministering the gene is by accelerating it into the desired cells as acoating on particles or otherwise.

A further aspect of the invention relates to an immunologicalcomposition which, when introduced into a host capable of having inducedwithin it an immunological response, induces an immunological responsein such host, wherein the composition comprises recombinant DNA whichcodes for and expresses an antigen of the hyperimmune serum reactiveantigens and fragments thereof of the present invention. Theimmunological response may be used therapeutically or prophylacticallyand may take the form of antibody immunity or cellular immunity such asthat arising from CTL or CD4+ T cells.

The hyperimmune serum reactive antigens and fragments thereof of theinvention or a fragment thereof may be fused with a co-protein which maynot by itself produce antibodies, but is capable of stabilizing thefirst protein and producing a fused protein which will have immunogenicand protective properties. This fused recombinant protein preferablyfurther comprises an antigenic co-protein, such asGlutathione-S-transferase (GST) or beta-galactosidase, relatively largeco-proteins which solubilise the protein and facilitate production andpurification thereof. Moreover, the co-protein may act as an adjuvant inthe sense of providing a generalized stimulation of the immune system.The co-protein may be attached to either the amino or carboxy terminusof the first protein.

Also, provided by this invention are methods using the described nucleicacid molecule or particular fragments thereof in such geneticimmunization experiments in animal models of infection with H. pylori.Such fragments will be particularly useful for identifying proteinepitopes able to provoke a prophylactic or therapeutic immune response.This approach can allow for the subsequent preparation of monoclonalantibodies of particular value from the requisite organ of the animalsuccessfully resisting or clearing infection for the development ofprophylactic agents or therapeutic treatments of H. pylori infection inmammals, particularly humans.

The hyperimmune serum reactive antigens and fragments thereof may beused as an antigen for vaccination of a host to produce specificantibodies which protect against invasion of bacteria, for example byblocking adherence of bacteria to damaged tissue. Examples of tissuedamage include wounds in skin or connective tissue caused e.g. bymechanical, chemical or thermal damage or by implantation of indwellingdevices, or wounds in the mucous membranes, such as the mouth, mammaryglands, urethra or vagina.

The present invention also includes a vaccine formulation, whichcomprises the immunogenic recombinant protein together with a suitablecarrier. Since the protein may be broken down in the stomach, it ispreferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous,intradermal intranasal or transdermal. Formulations suitable forparenteral administration include aqueous and non-aqueous sterileinjection solutions which may contain anti-oxidants, buffers,bacteriostats and solutes which render the formulation isotonic with thebodily fluid, preferably the blood, of the individual; and aqueous andnon-aqueous sterile suspensions which may include suspending agents orthickening agents. The formulations may be presented in unit-dose ormulti-dose containers, for example, sealed ampoules and vials, and maybe stored in a freeze-dried condition requiring only the addition of thesterile liquid carrier immediately prior to use. The vaccine formulationmay also include adjuvant systems for enhancing the immunogenicity ofthe formulation, such as oil-in-water systems and other systems known inthe art. The dosage will depend on the specific activity of the vaccineand can be readily determined by routine experimentation.

According to another aspect, the present invention relates to apharmaceutical composition comprising such a hyperimmune serum-reactiveantigen or a fragment thereof as provided in the present invention forH. pylori. Such a pharmaceutical composition may comprise one,preferably at least two or more hyperimmune serum reactive antigens orfragments thereof against H. pylori. Optionally, such H. pylorihyperimmune serum reactive antigens or fragments thereof may also becombined with antigens against other pathogens in a combinationpharmaceutical composition. Preferably, said pharmaceutical compositionis a vaccine for preventing or treating an infection caused by H. pyloriand/or other pathogens against which the antigens have been included inthe vaccine.

According to a further aspect, the present invention relates to apharmaceutical composition comprising a nucleic acid molecule encoding ahyperimmune serum-reactive antigen or a fragment thereof as identifiedabove for H. pylori Such a pharmaceutical composition may comprise oneor more nucleic acid molecules encoding hyperimmune serum reactiveantigens or fragments thereof against H. pylori. Optionally, such H.pylori nucleic acid molecules encoding hyperimmune serum reactiveantigens or fragments thereof may also be combined with nucleic acidmolecules encoding antigens against other pathogens in a combinationpharmaceutical composition. Preferably, said pharmaceutical compositionis a vaccine for preventing or treating an infection caused by H. pyloriand/or other pathogens against which the antigens have been included inthe vaccine.

The pharmaceutical composition may contain any suitable auxiliarysubstances, such as buffer substances, stabilisers or further activeingredients, especially ingredients known in connection ofpharmaceutical composition and/or vaccine production.

A preferable carrier/or excipient for the hyperimmune serum-reactiveantigens, fragments thereof or a coding nucleic acid molecule thereofaccording to the present invention is an immunostimulatory compound forfurther stimulating the immune response to the given hyperimmuneserum-reactive antigen, fragment thereof or a coding nucleic acidmolecule thereof. Preferably the immunostimulatory compound in thepharmaceutical preparation according to the present invention isselected from the group of polycationic substances, especiallypolycationic peptides, immunostimulatory nucleic acids molecules,preferably immunostimulatory deoxynucleotides, alum, Freund's completeadjuvants, Freund's incomplete adjuvants, neuroactive compounds,especially human growth hormone, or combinations thereof.

It is also within the scope of the present invention that thepharmaceutical composition, especially vaccine, comprises apart from thehyperimmune serum reactive antigens, fragments thereof and/or codingnucleic acid molecules thereof according to the present invention othercompounds which are biologically or pharmaceutically active. Preferably,the vaccine composition comprises at least one polycationic peptide. Thepolycationic compound(s) to be used according to the present inventionmay be any polycationic compound, which shows the characteristic effectsaccording to the WO 97/30721. Preferred polycationic compounds areselected from basic polyppetides, organic polycations, basic polyaminoacids or mixtures thereof. These polyamino acids should have a chainlength of at least 4 amino acid residues (WO 97/30721). Especiallypreferred are substances like polylysine, polyarginine and polypeptidescontaining more than 20%, especially more than 50% of basic amino acidsin a range of more than 8, especially more than 20, amino acid residuesor mixtures thereof. Other preferred polycations and theirpharmaceutical compositions are described in WO 97/30721 (e.g.polyethyleneimine) and WO 99/38528. Preferably these polypeptidescontain between 20 and 500 amino acid residues, especially between 30and 200 residues.

These polycationic compounds may be produced chemically or recombinantlyor may be derived from natural sources.

Cationic (poly)peptides may also be anti-microbial with properties asreviewed in {Ganz, T., 1999}. These (poly)peptides may be of prokaryoticor animal or plant origin or may be produced chemically or recombinantly(WO 02/13857). Peptides may also belong to the class of defensins (WO02/13857). Sequences of such peptides can be, for example, found in theAntimicrobial Sequences Database under the following internet address:

-   -   http://www.bbcm.univ.trieste.it/˜tossi/pag2.html

Such host defence peptides or defensives are also a preferred form ofthe polycationic polymer according to the present invention. Generally,a compound allowing as an end product activation (or down-regulation) ofthe adaptive immune system, preferably mediated by APCs (includingdendritic cells) is used as polycationic polymer.

Especially preferred for use as polycationic substances in the presentinvention are cathelicidin derived antimicrobial peptides or derivativesthereof (International patent application WO 02/13857, incorporatedherein by reference), especially antimicrobial peptides derived frommammalian cathelicidin, preferably from human, bovine or mouse.

Polycationic compounds derived from natural sources include HIV-REV orHIV-TAT (derived cationic peptides, antennapedia peptides, chitosan orother derivatives of chitin) or other peptides derived from thesepeptides or proteins by biochemical or recombinant production. Otherpreferred polycationic compounds are cathelin or related or derivedsubstances from cathelin. For example, mouse cathelin is a peptide,which has the amino acid sequenceNH₂-RLAGLLRKGGEKIGEKLKKIGOKIKNFFQKLVPQPE-COOH. Related or derivedcathelin substances contain the whole or parts of the cathelin sequencewith at least 15-20 amino acid residues. Derivations may include thesubstitution or modification of the natural amino acids by amino acids,which are not among the 20 standard amino acids. Moreover, furthercationic residues may be introduced into such cathelin molecules. Thesecathelin molecules are preferred to be combined with the antigen. Thesecathelin molecules surprisingly have turned out to be also effective asan adjuvant for an antigen without the addition of further adjuvants. Itis therefore possible to use such cathelin molecules as efficientadjuvants in vaccine formulations with or without furtherimmunactivating substances.

Another preferred polycationic substance to be used according to thepresent invention is a synthetic peptide containing at least 2KLK-motifs separated by a linker of 3 to 7 hydrophobic amino acids(International patent application WO 02/32451, incorporated herein byreference).

The pharmaceutical composition of the present invention may furthercomprise immunostimulatory nucleic acid(s). Immunostimulatory nucleicacids are e.g. neutral or artificial CpG containing nucleic acids, shortstretches of nucleic acids derived from non-vertebrates or in form ofshort oligonucleotides (ODNs) containing non-methylated cytosine-guaninedi-nucleotides (CpG) in a certain base context (e.g. described in WO96/02555). Alternatively, also nucleic acids based on inosine andcytidine as e.g. described in the WO 01/93903, or deoxynucleic acidscontaining deoxy-inosine and/or deoxyuridine residues (described in WO01/93905 and PCT/EP 02/05448, incorporated herein by reference) maypreferably be used as immunostimulatory nucleic acids for the presentinvention. Preferablly, the mixtures of different immunostimulatorynucleic acids may be used according to the present invention.

It is also within the present invention that any of the aforementionedpolycationic compounds is combined with any of the immunostimulatorynucleic acids as aforementioned. Preferably, such combinations areaccording to the ones as described in WO 01/93905, WO 02/32451, WO01/54720, WO 01/93903, WO 02/13857 and PCT/EP 02/05448 and the Austrianpatent application A 1924/2001, incorporated herein by reference.

In addition or alternatively such vaccine composition may comprise apartfrom the hyperimmune serum reactive antigens and fragments thereof, andthe coding nucleic acid molecules thereof according to the presentinvention a neuroactive compound. Preferably, the neuroactive compoundis human growth factor as, e.g. described in WO 01/24822. Alsopreferably, the neuroactive compound is combined with any of thepolycationic compounds and/or immunostimulatory nucleic acids asafore-mentioned.

In a further aspect the present invention is related to a pharmaceuticalcomposition. Such pharmaceutical composition is, for example, thevaccine described herein. Also a pharmaceutical composition is apharmaceutical composition which comprises any of the followingcompounds or combinations thereof: the nucleic acid molecules accordingto the present invention, the hyperimmune serum reactive antigens andfragments thereof according to the present invention, the vectoraccording to the present invention, the cells according to the presentinvention, the antibody according to the present invention, thefunctional nucleic acids according to the present invention and thebinding peptides such as the anticalines according to the presentinvention, any agonists and antagonists screened as described herein. Inconnection therewith any of these compounds may be employed incombination with a non-sterile or sterile carrier or carriers for usewith cells, tissues or organisms, such as a pharmaceutical carriersuitable for administration to a subject. Such compositions comprise,for instance, a media additive or a therapeutically effective amount ofa hyperimmune serum reactive antigen and fragments thereof of theinvention and a pharmaceutically acceptable carrier or excipient. Suchcarriers may include, but are not limited to, saline, buffered saline,dextrose, water, glycerol, ethanol and combinations thereof. Theformulation should suit the mode of administration.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical application,for example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.05-5 μg antigen/per kg of body weight,and such dose is preferably administered 1-3 times and with an intervalof 1-3 weeks.

With the indicated dose range, no adverse toxicological effects shouldbe observed with the compounds of the invention, which would precludetheir administration to suitable individuals.

In a further embodiment the present invention relates to diagnostic andpharmaceutical packs and kits comprising one or more containers filledwith one or more of the ingredients of the aforementioned compositionsof the invention. The ingredient(s) can be present in a useful amount,dosage, formulation or combination. Associated with such container(s)can be a notice in the form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biologicalproducts, reflecting approval by the agency of the manufacture, use orsale of the product for human administration.

In connection with the present invention any disease related use asdisclosed herein such as, e.g. use of the pharmaceutical composition orvaccine, is particularly a disease or diseased condition which is causedby, linked or associated with Helicobacter, more preferably, H. pylori.In connection therewith it is to be noted that H. pylori comprisesseveral strains including those disclosed herein. A disease related,caused or associated with the bacterial infection to be prevented and/ortreated according to the present invention includes besides otherspeptic ulcer and associated cancer in humans.

In a still further embodiment the present invention is related to ascreening method using any of the hyperimmune serum reactive antigens ornucleic acids according to the present invention. Screening methods assuch are known to the one skilled in the art and can be designed suchthat an agonist or an antagonist is screened. Preferably an antagonistis screened which in the present case inhibits or prevents the bindingof any hyperimmune serum reactive antigen and fragment thereof accordingto the present invention to an interaction partner. Such interactionpartner can be a naturally occurring interaction partner or anon-naturally occurring interaction partner.

The invention also provides a method of screening compounds to identifythose, which enhance (agonist) or block (antagonist) the function ofhyperimmune serum reactive antigens and fragments thereof or nucleicacid molecules of the present invention, such as its interaction with abinding molecule. The method of screening may involve high-throughput.

For example, to screen for agonists or antagonists, the interactionpartner of the nucleic acid molecule and nucleic acid, respectively,according to the present invention, maybe a synthetic reaction mix, acellular compartment, such as a membrane, cell envelope or cell wall, ora preparation of any thereof, may be prepared from a cell that expressesa molecule that binds to the hyperimmune serum reactive antigens andfragments thereof of the present invention. The preparation is incubatedwith labelled hyperimmune serum reactive antigens and fragments thereofin the absence or the presence of a candidate molecule, which may be anagonist or antagonist. The ability of the candidate molecule to bind thebinding molecule is reflected in decreased binding of the labelledligand. Molecules which bind gratuitously, i.e., without inducing thefunctional effects of the hyperimmune serum reactive antigens andfragments thereof, are most likely to be good antagonists. Moleculesthat bind well and elicit functional effects that are the same as orclosely related to the hyperimmune serum reactive antigens and fragmentsthereof are good agonists.

The functional effects of potential agonists and antagonists may bemeasured, for instance, by determining the activity of a reporter systemfollowing interaction of the candidate molecule with a cell orappropriate cell preparation, and comparing the effect with that of thehyperimmune serum reactive antigens and fragments thereof of the presentinvention or molecules that elicit the same effects as the hyperimmuneserum reactive antigens and fragments thereof. Reporter systems that maybe useful in this regard include but are not limited to colorimetriclabelled substrate converted into product, a reporter gene that isresponsive to changes in the functional activity of the hyperimmuneserum reactive antigens and fragments thereof, and binding assays knownin the art.

Another example of an assay for antagonists is a competitive assay thatcombines the hyperimmune serum reactive antigens and fragments thereofof the present invention and a potential antagonist with membrane-boundbinding molecules, recombinant binding molecules, natural substrates orligands, or substrate or ligand mimetics, under appropriate conditionsfor a competitive inhibition assay. The hyperimmune serum reactiveantigens and fragments thereof can be labelled such as by radioactivityor a colorimetric compound, such that the molecule number of hyperimmuneserum reactive antigens and fragments thereof bound to a bindingmolecule or converted to product can be determined accurately to assessthe effectiveness of the potential antagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a hyperimmune serum reactiveantigen and fragments thereof of the invention and thereby inhibit orextinguish its acitivity. Potential antagonists also may be smallorganic molecules, a peptide, a polypeptide such as a closely relatedprotein or antibody that binds to the same sites on a binding moleculewithout inducing functional activity of the hyperimmune serum reactiveantigens and fragments thereof of the invention.

Potential antagonists include a small molecule, which binds to andoccupies the binding site of the hyperimmune serum reactive antigens andfragments thereof thereby preventing binding to cellular bindingmolecules, such that normal biological activity is prevented. Examplesof small molecules include but are not limited to small organicmolecules, peptides or peptide-like molecules.

Other potential antagonists include antisense molecules (see {Okano, H.et al., 1991}; OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENEEXPRESSION; CRC Press, Boca Ration, Fla. (1988), for a description ofthese molecules).

Preferred potential antagonists include derivatives of the hyperimmuneserum reactive antigens and fragments thereof of the invention.

As used herein the activity of a hyperimmune serum reactive antigen andfragment thereof according to the present invention is its capability tobind to any of its interaction partner or the extent of such capabilityto bind to its or any interaction partner.

In a particular aspect, the invention provides the use of thehyperimmune serum reactive antigens and fragments thereof, nucleic acidmolecules or inhibitors of the invention to interfere with the initialphysical interaction between a pathogen and mammalian host responsiblefor sequelae of infection. In particular the molecules of the inventionmay be used: i) in the prevention of adhesion of H. pylori to mammalianextracellular matrix proteins on in-dwelling devices or to extracellularmatrix proteins in mucosal wounds; ii) to block protein mediatedmammalian cell invasion by, for example, initiating phosphorylation ofmammalian tyrosine kinases {Rosenshine, I. et al., 1992} to blockbacterial adhesion between mammalian extracellular matrix proteins andbacterial proteins which mediate tissue damage; iv) to block the normalprogression of pathogenesis in infections initiated other than by theimplantation of in-dwelling devices or by other surgical techniques.

Each of the DNA coding sequences provided herein may be used in thediscovery and development of antibacterial compounds. The encodedprotein upon expression can be used as a target for the screening ofantibacterial drugs. Additionally, the DNA sequences encoding the aminoterminal regions of the encoded protein or Shine-Delgarno or othertranslation facilitating sequences of the respective mRNA can be used toconstruct antisense sequences to control the expression of the codingsequence of interest.

The antagonists and agonists may be employed, for instance, to inhibitdiseases arising from infection with Helicobacter, especially H. pylori,such as peptic ulcer disease and gastric cancer.

In a still further aspect the present invention is related to anaffinity device such affinity device comprises as least a supportmaterial and any of the hyperimmune serum reactive antigens andfragments thereof according to the present invention, which is attachedto the support material. Because of the specificity of the hyperimmuneserum reactive antigens and fragments thereof according to the presentinvention for their target cells or target molecules or theirinteraction partners, the hyperimmune serum reactive antigens andfragments thereof allow a selective removal of their interactionpartner(s) from any kind of sample applied to the support materialprovided that the conditions for binding are met. The sample may be abiological or medical sample, including but not limited to, fermentationbroth, cell debris, cell preparation, tissue preparation, organpreparation, blood, urine, lymph liquid, liquor and the like.

The hyperimmune serum reactive antigens and fragments thereof may beattached to the matrix in a covalent or non-covalent manner. Suitablesupport material is known to the one skilled in the art and can beselected from the group comprising cellulose, silicon, glass, aluminium,paramagnetic beads, starch and dextrane.

The present invention is further illustrated by the following figures,examples and the sequence listing from which further features,embodiments and advantages may be taken. It is to be understood that thepresent examples are given by way of illustration only and not by way oflimitation of the disclosure.

In connection with the present invention

FIG. 1 shows the characterization of H. pylori specific human sera.

FIG. 2 shows the characterization of the small fragment genomic libraryLHP1-50 from H. pylori isolate KTH-Ca1.

FIG. 3 shows the characterization of the small fragment genomic libraryLHP2-50 from H. pylori isolate KTH-Du.

FIG. 4 shows the selection of bacterial cells by MACS using biotinylatedhuman IgGs.

Table 1 shows the summary of all screens performed with genomic H.pylori libraries and human serum.

Table 2 shows the summary of all gene distribution experiments performedwith genomic H. pylori DNA from individual isolates and gene specificoligonucleotides.

Table 3 shows the summary of epitope serology analysis with human sera.

The figures to which it might be referred to in the specification aredescribed in the following in more details.

FIG. 1 shows the characterization of human sera for anti-H. pyloriantibodies as measured by immune assays. Total anti-H. pylori IgG andIgA antibody levels were measured by standard ELISA using totalbacterial lysate prepared from H. pylori KTH Ca1 strain as coatingantigen. (A) Serum samples from randomly selected 54 adults wereanalysed for antibody levels and afterwards interviewed for symptoms(gasctric pain) and previous history of H. pylori infections. Fourindividuals were identified with high antibody titers without symptomsor disease (S−) and four with acute symptoms or known clinical disease(S+). (B) Sera from patients presenting themselves with typical symptomsof H. pylori diseases were analysed for IgG and IgA levels and groupedbased on negative or positive results in the Urease test performed byclinicians. ELISA units are calculated from absorbance readings at twoserum dilutions (10.000× and 50.000×). Averages for the two differentgroups are given. (C) Immunoblot analysis was performed on serapre-selected by ELISA in order to ensure multiple immune reactivity withprotein antigens. Results of a representative experiment using totalbacterial lysate prepared from H. pylori KTH Ca1 strain and selectedsera at 5.000× dilution are shown. Serum samples are from; Lane 1: ahigh titer S-individual, Lane 2: a high titer urease-patient, Lane 3 and4: a high titer urease+patient. Lane 1-3 was developed with anti-humanIgG secondary antibody and Lane 4 with anti-human IgA secondaryantibody. Mw: molecular weigth markers.

FIG. 2 shows the fragment size distribution of the KTH-Ca1 H. pyloristrain small fragment genomic library, LHP1-50. After sequencing 576randomly selected clones sequences were trimmed to eliminate vectorresidues and the number of clones with various genomic fragment sizeswere plotted. (B) Graphic illustration of the distribution of the sameset of randomly sequenced clones of LHP2-50 over the H. pylorichromosome. Blue circles indicate matching sequences to annotated ORFsin both +/+ and +/− orientations. Red rectangles represent fully matchedclones to non-coding chromosomal sequences in both +/+ and +/−orientations. Green diamonds positions all clones with chimericsequences. Numeric distances in base pairs are indicated over eachcircular genome for orientation. Partitioning of various clone setswithin the library is given in numbers and percentage at the bottom ofthe figure.

FIG. 3 shows the fragment size distribution of the KTH-Du H. pyloristrain small fragment genomic library, LHP2-50. After sequencing 576randomly selected clones sequences were trimmed to eliminate vectorresidues and the number of clones with various genomic fragment sizeswere plotted. (B) Graphic illustration of the distribution of the sameset of randomly sequenced clones of LHP2-50 over the H. pylorichromosome. Blue circles indicate matching sequences to annotated ORFsin both +/+ and +/− orientations. Red rectangles represent fully matchedclones to non-coding chromosomal sequences in both +/+ and +/−orientations. Green diamonds positions all clones with chimericsequences. Numeric distances in base pairs are indicated over eachcircular genome for orientation. Partitioning of various clone setswithin the library is given in numbers and percentage at the bottom ofthe figure.

FIG. 4A shows the MACS selection with biotinylated human IgGs. TheLHP1-50 library in pMAL9.1 was screened with 10 μg biotinylated, humanserum (P6-IgG) in the first and with 10 μg in the second selectionround. As negative control, no serum was added to the library cells forscreening. Number of cells selected after the 1^(st) and 2^(nd) elutionare shown for each selection round. FIG. 4B shows the reactivity ofspecific clones (1-26) isolated by bacterial surface display as analysedby Western blot analysis with the human serum (P6-IgG) used forselection by MACS at a dilution of 1:3,000. As a loading control thesame blot was also analysed with antibodies directed against theplatform protein LamB at a dilution of 1:5,000. LB, Extract from a doneexpressing LamB without foreign peptide insert.

Table 1: Immunogenic Proteins Identified by Bacterial Surface Display.

A, 300 bp library of H. pylori KTH Ca1 in fhuA with IC7-IgG (757), B,300 bp library of H. pylori KTH Ca1 in fhuA with P5-IgG (729), C, 300 bplibrary of H. pylori KTH Ca1 in fhuA with P9-IgG (441), D, 50 bp libraryof H. pylori KTH Ca1 in lamB with IC7-IgG (448), E, 50 bp library of H.pylori KTH Ca1 in lamB with P5-IgA (1130), F, 50 bp library of H. pyloriKTH Ca1 in lamB with P5-IgG (911), G, 50 bp library of H. pylori KTH Ca1in lamB with P6-IgA (1135), H, 50 bp library of H. pylori KTH Ca1 inlamB with P6-IgG (844), I, 50 bp library of H. pylori KTH Ca1 in lamBwith P9-IgG (1121), J, 300 bp library of H. pylori KTH Du in fhuA withP6-IgG (433), K, 300 bp library of H. pylori KTH Du in fhuA with P8-IgG(550), L, 50 bp library of H. pylori KTH Du in lamB with P6-IgG (1077),M, 50 bp library of H. pylori KTH Du in lamB with P8-IgG (740); *,prediction of antigenic sequences longer than 5 amino acids wasperformed with the program ANTIGENIC {Kolaskar, A. et al., 1990}.

Table 2: Gene Distribution in H. pylori strains.

28 H. pylori strains (including H. pylori KTH-Ca1 and KTH-Du, seeexample 2) and were tested by PCR with oligonucleotides specific for thegenes encoding relevant antigens. The PCR fragment of one selected PCRfragment was sequenced in order to confirm the amplification of thecorrect DNA fragment. *, number of amino acid substitutions in a H.pylori strain derived from a cancer patient as compared to H. pyloristrain KE26695 (aa, amino acids). #, an alternative strain was used forsequencing.

Table 3: Epitope Serology with Human Sera.

Immune reactivity of individual synthetic peptides representing selectedepitopes with individual human sera is shown. Extent of reactivity iscolour coded; white: neg (<500), light grey: + (500-650), dark grey: ++(650-800), and black: +++ (>800). Numbers represent ELISA readingsgenerated by measuring OD_(405nm) at serum dilution 200×. S stands forscore, calculated as the sum of all reactivities (addition of the numberof all +); P1 to P18 sera are from patients with definitive clinicaldiagnosis of duodenal or gastric ulcer, N1-4 sera are from healthyadults with high anti-H. pylori antibody titers without clinicalsymptomes of H. pylori disease. Location of synthetic peptides withinthe antigenic ORFs according to the genome annotation of H. pyloristrain 26695 are given in columns from and to indicating the first andlast amino acid residues, respectively. Peptide names: HP0009.1 presentin annotated ORF HP0009.

EXAMPLES Example 1 Characterization and Selection of Human Sera BasedAnti-H. pylori Antibodies, Preparation of Antibody Screening Reagents

Experimental Procedures

Enzyme Linked Immune Assay (ELISA).

ELISA plates (Maxisorb, Millipore) were coated with 5-10 μg/ml totalprotein diluted in coating buffer (0.1M sodium carbonate pH 9.2). Threedilutions of sera (2,000×, 10,000×, 50,000×) were made in PBS, BSA.Highly specific Horse Radish Peroxidase (HRP)-conjugated anti-human IgGor anti-human IgA secondary antibodies (Southern Biotech) were usedaccording to the manufacturers' recommendations (dilution: 1,000×).Antigen-antibody complexes were quantified by measuring the conversionof the substrate (ABTS) to colored product based on OD_(405nm) readingsby automatic ELIAS reader (TECAN SUNRISE).

Preparation of Bacterial Antigen Extracts

H. pylori KTH DU or KTH Ca1 strains were grown for 48 hours on agarplates, cells collected and lysed by repeated freeze-thaw cycles:incubation on dry ice/ethanol-mixture until frozen (1 min), then thawedat 37° C. (5 min): repeated 3 times. This was followed by sonication andcollection of supernatant by centrifugation (3,500 rpm, 15 min, 4° C.).

Immunoblotting

Total bacterial lysate preparations were prepared from in vitro grown H.pylori KTH DU or KTH Ca1 strains. 10 to 25 μg total protein/lane wasseparated by SDS-PAGE using the BioRad Mini-Protean 3 Cellelectrophoresis system and proteins transferred to nitrocellulosemembrane (ECL, Amersham Pharmacia). After overnight blocking in 5% milk,human sera were added at 2,000× dilution, and HRPO labeled anti-humanIgG was used for detection.

Purification of antibodies for genomic screening. Five sera from boththe patient and the healthy group were selected based on the overallanti-streptococcal titers for a serum pool used in the screeningprocedure. Antibodies against E. coli proteins were removed byincubating the heat-inactivated sera with whole cell E. coli cells(DH5alpha, transformed with pHIE11, grown under the same condition asused for bacterial surface display). Highly enriched preparations ofIgGs from the pooled, depleted sera were generated by protein G affinitychromatography, according to the manufacturer's instructions (UltraLinkImmobilized Protein G, Pierce). IgA antibodies were purified also byaffinity chromatography using biotin-labeled anti-human IgA (SouthernBiotech) immobilized on Streptavidin-agarose (GIBCO BRL). The efficiencyof depletion and purification was checked by SDS-PAGE, Western blotting,ELISA and protein concentration measurements.

Results

The antibodies produced against H. pylori by the human immune system andpresent in human sera are indicative of the in vivo expression of theantigenic proteins and their immunogenicity. These molecules areessential for the identification of individual antigens in the approachas described in the present invention, which is based on the interactionof the specific antibodies and the corresponding H. pylori peptides orproteins. To gain access to relevant antibody repertoires, human serawere collected from

-   -   I. 54 randomly selected healthy adults. Individuals were        interviewed for the presence or absence of clinical symptoms and        previously diagnosed H. pylori infection.    -   II. patients with duodenal ulcer.    -   III. patients with gastric ulcer and cancer.        For the patient groups H. pylori infection was confirmed and        medical diagnosis was based on medical microbiological tests,        Urease test, biopsy or gastroscopy. A total of 191 sera from        patients were included in the analysis.

The sera were characterized for anti-H. pylori antibodies by a series ofELISA and immunoblotting assays.

For that purpose two different antigen preparation were used: whole cellextracts prepared from H. pylori strains KTH-Ca1 and KTH-Du and both IgGand IgA antibody levels were determined. Antibody titers were expressedas units calculated from absorbance readings at two differentdilutions—10.000× and 50.000× for IgG and 1.000× and 5.000× forIgA—where the response was linear (FIGS. 1A and B). Among the high titerrandomly taken individuals eigth out of the 54 included (15%) showedsignificant IgG and IgA antibody levels. Half of these individuals wereknown H. pylori ‘patients’ acutely or before, the other half had nomedical history or any complains. Sera of these four individuals werepooled and prepared for antigen identification. Since H. pyloriinfections are common, antibodies are present as a consequence ofnatural immunization from previous encounters with Helicobacter evenwithout consequent carriage. The value of the ELISA assay imployed werefurther proved by analyzing patients' sera with or without activedisease. Comparing the antibody levels in urease test positive andurease test negative individuals, significantly higher antibody levelswere measured in the Urease+group (FIG. 1B). According to literaturedata, the false negative cases (˜10%) are much more prevalent then thefalse positives in this test, suggesting that the ELIAS assays is likelyto be even more powerful predicting active H. pylori infections. Serawere ranked based on the reactivity against total lysate preparation inboth antibody classes, and the highest ones from all three serum donorgroups were selected for further analysis by immunoblotting. This latterassay confirmed immune reactivity against multiple H. pylori proteins,as it is examplified on FIG. 1C.

This extensive antibody characterization approach has led to theunambiguous identification of anti-Helicobacter hyperimmune sera andallowed the preparation of 5 donor pools.

Example 2 Generation of Highly Random, Frame-Selected, Small-Fragment,Genomic DNA Libraries of Helicobacter pylori

Experimental Procedures

Preparation of helicobacter genomic DNA. Sufficient amounts of bacterialpellets from the KTH-Ca1 and KTH-Du clinical isolates of H. pylori wereobtained from Dr. Lars Engstrand. Bacterial pellets were washed 3× withPBS and carefully re-suspended in 0.5 ml of Lysozyme solution (100mg/ml). 0.1 ml of 10 mg/ml heat treated RNase A and 20 U of RNase T1were added, mixed carefully and the solution was incubated for 1 h at37° C. Following the addition of 0.2 ml of 20% SDS solution and 0.1 mlof Proteinase K (10 mg/ml) the tube was incubated overnight at 55° C.1/3 volume of saturated NaCl was then added and the solution wasincubated for 20 min at 4° C. The extract was pelleted in a microfuge(13,000 rpm) and the supernatant transferred into a new tube. Thesolution was extracted with PhOH/CHCl₃/IAA (25:24:1) and with CHCl₃/IAA(24:1). DNA was precipitated at room temperature by adding 0.6× volumeof Isopropanol, spooled from the solution with a sterile Pasteur pipetteand transferred into tubes containing 80% ice-cold ethanol. DNA wasrecovered by centrifuging the precipitates with 10-12,000×g, then driedon air and dissolved in ddH₂O.

Preparation of small genomic DNA fragments. Genomic DNA was mechanicallysheared into fragments ranging in size between 150 and 300 bp using acup-horn sonicator (Bandelin Sonoplus UV 2200 sonicator equipped with aBB5 cup horn, 10 sec. pulses at 100% power output) or into fragments ofsize between 50 and 70 bp by mild DNase I treatment (Novagen). It wasobserved that sonication yielded a much tighter fragment sizedistribution when breaking the DNA into fragments of the 150-300 bp sizerange. However, despite extensive exposure of the DNA to ultrasonicwave-induced hydromechanical shearing force, subsequent decrease infragment size could not be efficiently and reproducibly achieved.Therefore, fragments of 50 to 70 bp in size were obtained by mild DNaseI treatment using Novagen's shotgun cleavage kit. A 1:20 dilution ofDNase I provided with the kit was prepared and the digestion wasperformed in the presence of MnCl₂ in a 60 μl volume at 20° C. for 5 minto ensure double-stranded cleavage by the enzyme. Reactions were stoppedwith 2 μl of 0.5 M EDTA and the fragmentation efficiency was evaluatedon a 2% TAE-agarose gel. This treatment resulted in total fragmentationof genomic DNA into near 50-70 bp fragments. Fragments were thenblunt-ended twice using T4 DNA Polymerase in the presence of 100 μM eachof dNTPs to ensure efficient flushing of the ends. Fragments were usedimmediately in ligation reactions or frozen at −20° C. for subsequentuse.

Description of the vectors. The vector pMAL4.31 was constructed on apASK-IBA backbone {Skerra, k, 1994} with the beta-lactamase (bla) geneexchanged with the Kanamycin resistance gene. In addition, the bla genewas cloned into the multiple cloning site. The sequence encoding maturebeta-lactamase is preceded by the leader peptide sequence of ompA toallow efficient secretion across the cytoplasmic membrane. Furthermore asequence encoding the first 12 amino acids (spacer sequence) of maturebeta-lactamase follows the ompA leader peptide sequence to avoid fusionof sequences immediately after the leader peptidase cleavage site, sincee.g. clusters of positive charged amino acids in this region woulddecrease or abolish translocation across the cytoplasmic membrane{Kajava, A. et al., 2000}. A SmaI restriction site serves for libraryinsertion. An upstream FseI site and a downstream NotI site, which wereused for recovery of the selected fragment, flank the SmaI site. Thethree restriction sites are inserted after the sequence encoding the 12amino acid spacer sequence in such a way that the bla gene istranscribed in the −1 reading frame resulting in a stop codon 15 bpafter the NotI site. A +1 bp insertion restores the bla ORF so thatbeta-lactamase protein is produced with a consequent gain of Ampicillinresistance.

The vector pMAL9.1 was constructed by cloning the lamB gene into themultiple cloning site of pEH1 {Hashemzadeh-Bonehi, L. et al., 1998}.Subsequently, a sequence was inserted in lamB after amino acid 154,containing the restriction sites FseI, SmaI and NotI. The reading framefor this insertion was constructed in such a way that transfer offrame-selected DNA fragments excised by digestion with FseI and NotIfrom plasmid pMAL4.31 yields a continuous reading frame of lamB and therespective insert.

The vector pHIE11 was constructed by cloning the fhuA gene into themultiple cloning site of pEH1. Thereafter, a sequence was inserted infhuA after amino acid 405, containing the restriction site FseI, XbaIand NotI. The reading frame for this insertion was chosen in a way thattransfer of frame-selected DNA fragments excised by digestion with FseIand NotI from plasmid pMAL4.31 yields a continuous reading frame of fhuAand the respective insert.

Cloning and evaluation of the library for frame selection. Genomicfragments of H. pylori DNA were ligated into the SmaI site of the vectorpMAL4.31. Recombinant DNA was electroporated into DH10B electrocompetentE. coli cells (GIBCO BRL) and transformants plated on LB-agarsupplemented with Kanamycin (50 μg/ml) and Ampicillin (50 μg/ml). Plateswere incubated over night at 37° C. and colonies collected for largescale DNA extraction. A representative plate was stored and saved forcollecting colonies for colony PCR analysis and large-scale sequencing.A simple colony PCR assay was used to initially determine the roughfragment size distribution as well as insertion efficiency. Fromsequencing data the precise fragment size was evaluated, junctionintactness at the insertion site as well as the frame selection accuracy(3n+1 rule).

Cloning and evaluation of the library for bacterial surface display.Genomic DNA fragments were excised from the pMAL4.31 vector, containingthe H. pylori libraries with the restriction enzymes FseI and NotI. Theentire population of fragments was then transferred into plasmidspMAL9.1 (LamB) or pHIE11 (FhuA), which have been digested with FseI andNotI. Using these two restriction enzymes, which recognise an 8 bp GCrich sequence, the reading frame that was selected in the pMAL4.31vector is maintained in each of the platform vectors. The plasmidlibrary was then transformed into E. coli DH5alpha cells byelectroporation. Cells were plated onto large LB-agar platessupplemented with 50 μg/ml Kanamycin and grown over night at 37° C. at adensity yielding clearly visible single colonies. Cells were thenscraped off the surface of these plates, washed with fresh LB medium andstored in aliquots for library screening at −80° C.

Results

Libraries for frame selection. Four libraries (LHP1-50, LHP2-50,LHP1-300 and LHP2-300) were generated in the pMAL4.31 vector with sizesof approximately 50 and 300 bp, respectively. For each library, ligationand subsequent transformation of approximately 1 μg of pMAL4.31 plasmidDNA and 50 ng of fragmented genomic H. pylori DNA yielded 4×10⁵ to 2×10⁶clones after frame selection. To assess the randomness of the libraries,approximately 600 randomly chosen clones of LHP1-50 and LHP2-50 weresequenced. The bioinformatic analysis showed that of these clones onlyvery few were present more than once. Furthermore, it was shown forexample for LHP2-50 that 90% of the clones fell in the size rangebetween 19 and 64 bp with an average size of 28 bp (FIG. 2, 3). Allsequences followed the “3n+1 rule”, showing that all clones wereproperly frame selected.

Bacterial surface display libraries. The display of peptides on thesurface of E. coli required the transfer of the inserts from the LHPlibraries from the frame selection vector pMAL4.31 to the displayplasmids pMAL9.1 (LamB) or pHIE11 (FhuA). Genomic DNA fragments wereexcised by FseI and NotI restriction and ligation of 5 ng inserts with0.1 μg plasmid DNA and subsequent transformation into DH5alpha cellsresulted in 2-5×10⁶ clones. The clones were scraped off the LB platesand frozen without further amplification.

Example 3 Identification of Highly Immunogenic Peptide Sequences from H.pylori Using Bacterial Surface Displayed Genomic Libraries and HumanSerum

Experimental Procedures

MACS screening. Approximately 2.5×10⁸ cells from a given library weregrown in 5 ml LB-medium supplemented with 50 μg/ml Kanamycin for 2 h at37° C. Expression was induced by the addition of 1 mM IPTG for 30 min.Cells were washed twice with fresh LB medium and approximately 2×10⁷cells re-suspended in 100 μl LB medium and transferred to an Eppendorftube.

10 μg of biotinylated, human IgGs purified from serum was added to thecells and the suspension incubated over night at 4° C. with gentleshaking. 900 μl of LB medium was added, the suspension mixed andsubsequently centrifuged for 10 min at 6,000 rpm at 4° C. (for IgAscreens, 10 μg of purified IgAs were used and these captured withbiotinylated anti-human-IgA secondary antibodies). Cells were washedonce with 1 ml LB and then re-suspended in 100 μl LB medium. 10 μl ofMACS microbeads coupled to streptavidin (Miltenyi Biotech, Germany) wereadded and the incubation continued for 20 min at 4° C. Thereafter 900 μlof LB medium was added and the MACS microbead cell suspension was loadedonto the equilibrated MS column (Miltenyi Biotech, Germany) which wasfixed to the magnet. (The MS columns were equilibrated by washing oncewith 1 ml 70% EtOH and twice with 2 ml LB medium.)

The column was then washed three times with 3 ml LB medium. Afterremoval of the magnet, cells were eluted by washing with 2 ml LB medium.After washing the column with 3 ml LB medium, the 2 ml eluate was loadeda second time on the same column and the washing and elution processrepeated. The loading, washing and elution process was performed a thirdtime, resulting in a final eluate of 2 ml.

A second round of screening was performed as follows. The cells from thefinal eluate were collected by centrifugation and re-suspended in 1 mlLB medium supplemented with 50 μg/ml Kanamycin. The culture wasincubated at 37° C. for 90 min and then induced with 1 mM IPTG for 30min. Cells were subsequently collected, washed once with 1 ml LB mediumand suspended in 10 μl LB medium. Since the volume was reduced, 10 μg ofhuman, biotinylated IgGs was added and the suspension incubated overnight at 4° C. with gentle shaking. All further steps were exactly thesame as in the first selection round. Cells selected after two rounds ofselection were plated onto LB-agar plates supplemented with 50 μg/mlKanamycin and grown over night at 37° C.

Evaluation of selected clones by sequencing and Western blot analysis.Selected clones were grown over night at 37° C. in 3 ml LB mediumsupplemented with 50 μg/ml Kanamycin to prepare plasmid DNA usingstandard procedures. Sequencing was performed at MWG (Germany).

For Western blot analysis approximately 10 to 20 μg of total cellularprotein was separated by 10% SDS-PAGE and blotted onto HybondC membrane(Amersham Pharmacia Biotech, England). The LamB or FhuA fusion proteinswere detected using human serum as the primary antibody at a dilution ofapproximately 1:5,000 and anti-human IgG or IgA antibodies coupled toHRP at a dilution of 1:5,000 as secondary antibodies. Detection wasperformed using the ECL detection kit (Amersham Pharmacia Biotech,England). Alternatively, rabbit anti FhuA or mouse anti LamB antibodieswere used as primary antibodies in combination with the respectivesecondary antibodies coupled to HRP for the detection of the fusionproteins.

Results

Screening of bacterial surface display libraries by magnetic activatedcell sorting (MACS) using biotinylated Igs. The libraries LHP1-50 andLHP2-50 in pMAL9.1 and LHP1-300 and LHP2-300 in pHIE11 were screenedwith pools of biotinylated, human IgGs and IgAs from patient sera orsera from healthy individuals (see Example 1: Preparation of antibodiesfrom human serum). The selection procedure was performed as describedunder Experimental procedures. FIG. 4A shows a representative example ofa screen with the LHP1-50 library and P6-IgGs. As can be seen from thecolony count after the first selection cycle from MACS screening, thetotal number of cells recovered at the end is reduced from app. 1 to2×10⁷ cells to approximately 10⁴ cells, whereby the selection in thepresence of serum yielded a slightly higher number of cells than in theabsence of antibodies (FIG. 4A). After the second round of screeninghowever, a similar number of cells was recovered with P6-IgG, while onlya few hundred cells were recovered when no IgGs from human serum wereadded, clearly showing that selection was dependent on H. pylorispecific antibodies. To evaluate the performance of the screen,approximately 50 selected clones were picked randomly and subjected toWestern blot analysis with the same, pooled serum (FIG. 4B). Thisanalysis revealed that 70% of the selected clones showed reactivity withantibodies present in the relevant serum whereas the control strainexpressing LamB without a H. pylori specific insert did not react withthe same serum. In general, the rate of reactivity was observed to liewithin the range of 35 to 75%. Colony PCR analysis confirmed that allselected clones contained an insert in the expected size range.

Subsequent sequencing of a larger number of randomly picked clones (600to 1200 per screen) led to the identification of the gene and thecorresponding peptide or protein sequence that was specificallyrecognized by the human serum used for screening. The frequency withwhich a specific clone is selected reflects at least in part theabundance and/or affinity of the specific antibodies in the serum usedfor selection and recognizing the epitope presented by this done. Inthat regard it is striking that clones derived from some ORFs (e.g.HP0527, HP0547 and HP1341) were picked more than 100 times, indicatingtheir highly immunogenic property. Table 1 summarizes the data obtainedfor all 13 performed screens. All clones in Table 1 have been verifiedby Western blot analysis with whole cellular extracts from single clonesto show the indicated reactivity with the pool of human serum used inthe respective screen. As seen in Table 1, distinct regions of theidentified ORF are identified as immunogenic, since variably sizedfragments of the proteins are displayed on the surface by the platformproteins.

It is further worth noticing that most of the genes identified by thebacterial surface display screen encode proteins that are eitherattached to the surface of H. pylori and/or are secreted. This is inaccordance with the expected role of surface attached or secretedproteins in virulence of H. pylori.

Example 4 Gene Distribution Studies with Highly Immunogenic ProteinsIdentified from H. pylori

Experimental Procedures

Gene distribution of H. pylori antigens by PCR. In order to establishwhether the genes encoding the identified Helicobacter pylori antigensoccur ubiquitously in H. pylori strains, PCR was performed on a seriesof independent H. pylori isolates with primers specific for the gene ofinterest. H. pylori isolates were obtained from patients coveringvarious disease conditions associated with H. pylori infection (cancerpatients: 11 strains, duodenal ulcer: 6, atrophic gastritis: 5,gastritis: 3, Hiatus hernia: 1, normal controls: 2). Oligonucleotidesequences as primers were designed for all identified ORFs yieldingproducts of approximately 1,000 bp, if possible covering all identifiedimmunogenic epitopes. Genomic DNA of all H. pylori strains was preparedas described under Example 2. PCR was performed in a reaction volume of25 μl using Taq polymerase (1U), 200 nM dNTPs, 10 pMol of eacholigonucleotide and the kit according to the manufacturers instructions(Invitrogen, The Netherlands). As standard, 30 cycles (1×: 5 min. 95°C., 30×: 30 sec. 95° C., 30 sec. 56° C., 30 sec. 72° C., 1×4 min. 72°C.) were performed, unless conditions had to be adapted for individualprimer pairs.

Results

Identified genes encoding immunogenic proteins were tested by PCR fortheir presence in 28 different strains of H. pylori (Table 2). An idealvaccine antigen would be an antigen that is present in all, or the vastmajority of strains of the target organism to which the vaccine isdirected. For a large number of antigens, the PCR reaction amplified aDNA fragment of the correct size with all 28 chosen H. pylori strains(e.g. HP0563, HP0887, HP1341). The sequencing of one PCR fragment fromone individual strain showed that the amplified DNA fragment correspondsto the correct gene, but it also allows an estimation of the level ofvariation within this particular gene. While some genes possess acompletely identical amino acid sequence in the two compared strains(e.g. HP0121, HP0413, HP1374), most antigens showed some degree ofvariation as listed in Table 2. The sequencing revealed that the aminoacid sequences of approximately 80% of all antigens were to more than95% identical in the two analysed strains, with only 1 strain showing alevel of identity below 85%.

From a total of 106 genes analysed, 76 were present in all strainstested, 14 were present in more than 80% of the strains, while only 16genes were absent in more than 20% of the tested 28 strains (Table 2).In addition, only 9 genes (e.g. HP115, HP717, HP887, HP913 and HP1119)showed a clear variation in size but were present in all or most H.pylori isolates. Importantly, many of the identified antigens are wellconserved in all strains in sequence and size and are therefore novelvaccine candidates to prevent infections by H. pylori.

Example 5 Assessment of the Reactivity of Highly Immunogenic PeptideSequences from H. pylori with Individual Human Sera

Experimental Procedures

Peptide Synthesis

Peptides were synthesized in small scale (4 mg resin; up to 288 inparallel) using standard F-moc chemistry on a Rink amide resin (PepChem,Tübingen, Germany) using a SyroII synthesizer (Multisyntech, Witten,Germany). After the sequence was assembled, peptides were elongated withFmoc-epsilon-aminohexanoic acid (as a linker) and biotin (Sigma, St.Louis, Mo.; activated like a normal amino acid). Peptides were cleavedoff the resin with 93% TFA, 5% triethylsilane, and 2% water for onehour. Peptides were dried under vacuum and freeze dried three times fromacetonitrile/water (1:1). The presence of the correct mass was verifiedby mass spectrometry on a Reflex III MALDI-TOF (Bruker, Bremen Germany).The peptides were used without further purification.

Enzyme Linked Immune Assay (ELISA).

Biotin-labeled peptides (at the N-terminus) were coated on StreptavidinELISA plates (EXICON) at 10 μg/ml concentration according to themanufacturer's instructions. Highly specific Horse Radish Peroxidase(HRP)-conjugated anti-human IgG secondary antibodies (Southern Biotech)were used according to the manufacturers' recommendations (dilution:1,000×). Sera were tested at two serum dilutions, 200× and 1,000×.Following manual coating, peptide plates were processed and analyzed bythe Gemini 160 ELISA robot (TECAN) with a built-in ELISA reader (GENIOS,TECAN).

Results

Following the bioinformatic analysis of selected clones, correspondingpeptides were designed and synthesized. In case of epitopes with morethan 26 amino acid residues, overlapping peptides were made. Allpeptides were synthesized with a N-terminal biotin-tag and used ascoating reagents on Streptavidin-coated ELISA plates.

The analysis was performed with 144 peptides and 22 individual humanserum samples which were included in the serum pools used forpreparations of IgG and IgA screening reagents for bacterial surfacedisplay. A summary for serum reactivity of peptides representing H.pylori epitopes from the genomic screen analysed with human sera isshown in Table 3. The peptides were compared by the score calculated foreach peptide based on the number of positive sera and the extent ofreactivity. Peptides range from highly and widely reactive to weaklypositive ones. Among the most reactive ones there are known antigens,some of them are also protective in animal challenge models, such as theCagA (HP0547) and vacuolating cytotoxin (HP0887). Besides the knownantigens several novel highly immunogenic proteins and epitopes withinthose have been identified, such as the siderophore-mediated irontransport protein (HP1341), fumarate reductase flavoprotein subunit(frdA) (HP0192) and HP0087 hypothetical protein among others.

REFERENCES

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Tourdot, S., et al. (2000). Eur J Immunol 30: 3411-21. TABLE 1Immunogenic proteins identified by bacterial surface display. A, 300 bplibrary of H. pylori KTH Ca1 in fhuA with IC7-IgG (757), B, 300 bplibrary of H. pylori KTH Ca1 in fhuA with P5-IgG (729), C, 300 bplibrary of H. pylori KTH Ca1 in fhuA with P9-IgG (441), D, 50 bp libraryof H. pylori KTH Ca1 in lamB with IC7-IgG (448), E, 50 bp library of H.pylori KTH Ca1 in lamB with P5-IgA (1130), F, 50 bp library of H. pyloriKTH Ca1 in lamB with P5-IgG (911), G, 50 bp library of H. pylori KTH Ca1in lamB with P6- IgA (1135), H, 50 bp library of H. pylori KTH Ca1 inlamB with P6-IgG (844), I, 50 bp library of H. pylori KTH Ca1 in lamBwith P9-IgG (1121), J, 300 bp library of H. pylori KTH Du in fhuA withP6-IgG (433), K, 300 bp library of H. pylori KTH Du in fhuA with P8-IgG(550), L, 50 bp library of H. pylori KTH Du in lamB with P6-IgG (1077),M, 50 bp library of H. pylori KTH Du in lamB with P8-IgG (740); *,prediction of antigenic sequences longer than 5 amino acids wasperformed with the program ANTIGENIC (Kolaskar and Tongaonkar, 1990).No. of Location selected of identified H. pylori clones per im- Seq.antigenic Putative function ORF and munogenic ID (DNA, protein (byhomology) predicted immunogenic aa** screen region (aa) Prot.) HP0009outer membrane 63-91, 95-101, 110-116, 134-148, 150-156, 158-164, G: 1,H: 2, 239-308 1, 179 protein (omp1) 188-193, 197-209, 226-241, 247-254,291-297, 312-319, K: 2, M: 38 338-346, 351-358, 366-378, 404-410,420-438, 448-454, 465-473, 482-488, 490-498, 503-510, 512-519, 531-543,547-554, 568-575, 589-604, 610-631 HP0010 chaperone and heat 16-29,35-47, 50-68, 70-79, 91-101, 143-149, 158-163, K: 1 17-79, 2, 180 shockprotein 185-191, 196-206, 215-224, 230-237, 244-251, 258-278, 218-233(groEL) 290-311, 319-325, 338-351, 365-385, 396-429, 445-454, 458-466,491-499, 501-521 HP0043 mannose-6- 4-10, 16-41, 46-66, 77-84, 91-97,102-118, 125-144, 187-200, G: 1 460-470 3, 181 phosphate 202-214,245-253, 255-261, 286-295, 300-330, 335-342, isomerase (pmi) 350-361,363-381, 385-392, 396-416, 435-450 HP0063 hypothetical protein 11-19,27-48, 52-59, 77-82, 84-107, 118-125, 127-154, D: 4, E: 1, 313-331 4,182 178-183, 192-209, 215-221, 286-295, 302-313, 350-357, G: 2 402-415,417-431, 453-463, 465-493 HP0067 urease accessory 19-26, 30-43, 47-55,63-68, 72-80, 97-104, 107-119, 129-146, H: 1 26-43 5, 183 protein (ureH)160-175, 194-216, 231-251, 254-260 HP0072 urease beta subunit 7-13,29-37, 65-81, 110-120, 123-131, 135-152, 230-249, E: 2, G: 3, 533-567 6,184 (urea 254-260, 284-290, 292-299, 317-326, 329-336, 403-444, H: 1, M:2 amidohydrolase) 452-458, 466-477, 490-498, 510-519, 541-550, 557-566(ureB) HP0086 Conserved 5-47, 71-77, 79-86, 89-95, 120-126, 137-144,176-181, B: 3, C: 1, I: 4 59-75 7, 185 hypothetical protein 184-196,202-208, 211-232, 236-282, 301-313, 317-325, 341-347, 353-384, 394-400,412-433, 436-443 HP0087 hypothetical protein 4-18, 22-38, 59-69,106-112, 116-130, 138-149, 156-170, A: 2, B: 2, 1-104, 8, 186 175-197,200-214, 216-223, 233-244, 255-261, 266-276, E: 2, F: 1, 130-147279-286, 325-333, 342-348, 366-399, 402-420, 429-441 G: 2, I: 1, L: 2HP0088 RNA polymerase 50-58, 69-95, 97-113, 131-136, 157-163, 170-175,188-212, A: 2, B: 9, 35-46 9, 187 sigma-70 factor 220-226, 254-259,265-277, 283-289, 297-308, 311-318, D: 2, F: 3, (rpoD) 347-358, 360-369,378-401, 416-421, 440-450, 454-462, H: 4 470-476, 493-502, 506-514,536-567, 585-590, 598-607, 613-618, 653-659 HP0089 pfs protein (pfs)16-29, 32-60, 65-87, 89-123, 128-134, 137-158, 162-173, E: 11, G: 2206-225 10, 188 178-196, 210-216, 218-228 HP0115 flagellin B (flaB)10-20, 26-35, 51-64, 86-91, 94-100, 113-122, 154-160, F: 2, H: 1, 24-55,11, 189 185-191, 193-201, 211-217, 225-230, 237-246, 251-257, K: 1, L:52 236-341 298-304, 306-312, 316-328, 340-348, 357-389, 391-397,415-421, 449-456, 458-471, 488-495, 502-511 HP0175 cell binding factor 25-22, 41-51, 87-93, 114-122, 127-136, 150-156, 158-166, A: 11, C: 7,9-126, 12, 190 223-233, 245-263, 291-296 K: 2 127-285 HP0121phosphoenolpyruvate 30-43, 46-56, 61-70, 72-83, 85-93, 103-113, 119-125,H: 3, I: 4, K: 3 347-361 13, 191 synthase (ppsA) 151-166, 179-191,212-218, 225-231, 236-243, 262-267, 291-307, 331-344, 349-355, 366-372,380-386, 414-422, 428-447, 459-464, 469-478, 507-519, 525-544, 563-569,576-590, 620-626, 633-643, 654-659, 665-671, 684-707, 717-723, 725-733,747-779, 782-801 HP0123 threonyl-tRNA 4-12, 14-26, 37-80, 107-115,133-139, 144-150, 154-165, D: 6, F: 1, 7-23, 14, 192 synthetase (thrS)173-180, 191-199, 205-211, 221-231, 237-244, 254-284, H: 2, M: 1 465-479307-340, 342-353, 360-368, 370-380, 479-493, 495-503, 509-522, 525-536,539-547, 554-560, 565-573, 578-583 HP0130 hypothetical protein 4-17,47-55, 76-83, 85-100, 104-112, 117-123, 126-135, A: 1, C: 1, I: 258-28315, 193 142-148, 156-167, 174-182, 267-273 1, K: 1, M: 3 HP0150hypothetical protein 8-32, 36-42, 65-88, 102-108, 112-140, 147-163,170-179, A: 1, B: 1, 117-124 16, 194 183-193 D: 1, J: 1, L: 2 HP0183serine 12-18, 45-50, 62-77, 82-95, 99-113, 115-123, 125-147, I: 5333-350 17, 195 hydroxymethyltransferase 155-177, 187-209, 211-223,244-253, 259-270, 278-297, (glyA) 302-307, 311-318, 329-334, 350-356,359-365, 390-400, 402-413 HP0192 fumarate reductase, 4-13, 15-27, 30-46,53-58, 68-74, 82-95, 115-126, 134-139, H: 1, I: 4, 376-400 18, 196flavoprotein subunit 148-153, 159-176, 182-199, 201-217, 220-225,227-235, L: 20 (frdA) 237-248, 253-266, 300-315, 322-336, 390-396,412-426, 438-445, 448-459, 477-484, 502-508, 515-527, 529-537, 553-568,643-651, 658-667, 690-703 HP0197 S- 4-10, 24-32, 38-55, 59-67, 70-77,80-87, 89-97, 123-129, F: 1, H: 1, 73-94 19, 197 adenosylmethionine134-151, 166-172, 178-189, 191-216, 218-235, 245-259, L: 8 synthetase 2(metX) 271-315, 326-339, 341-360 HP0201 fatty 13-25, 31-38, 43-57,79-85, 92-99, 106-112, 117-128, L: 5 240-256 20, 198 acid/phospholipid130-139, 146-158, 160-175, 194-204, 211-222, 225-232, synthesis protein234-242, 263-270, 278-292, 299-320, 322-333 (plsX) HP0202beta-ketoacyl-acyl 4-17, 55-63, 66-101, 109-131, 135-143, 145-151,155-161, F: 1, I: 3 195-221 21, 199 carrier protein 164-170, 177-185,192-198, 213-218, 223-238, 246-256, synthase III 258-268, 273-283,309-314, 322-328 HP0210 chaperone and heat 13-24, 31-39, 41-50, 63-69,90-96, 104-109, 116-141, F: 1, M: 2 344-363 22, 200 shock protein C62.5148-153, 161-167, 173-178, 190-209, 253-258, 265-272, (htpG) 279-289,295-312, 317-343, 355-366, 376-389, 400-407, 430-451, 453-464, 466-472,487-493, 499-505, 523-538, 554-559, 568-579, 584-601 HP0211 conserved5-22, 30-36, 53-59, 61-70, 82-92, 99-106, 120-131, 135-148, A: 1, C: 1,111-249 23, 201 hypothetical 154-167, 169-183, 187-199, 204-212, 231-247K: 1 secreted protein HP0228 conserved 17-36, 40-66, 71-144, 148-171,173-191, 199-214, 220-255, A: 1, E: 6, 287-307 24, 202 hypothetical265-272, 278-288, 298-333, 342-385 F: 1 integral membrane protein HP0229outer membrane 4-16, 22-28, 30-36, 42-48, 95-116, 154-162, 164-174, E:1, F: 1, L: 33-44, 25, 203 protein (omp6) 239-252, 258-263, 273-285,306-313, 323-333, 341-357, 14 233-258, 363-369, 372-379, 395-401,430-436, 438-453, 464-480 349-369 HP0235 conserved 4-21, 30-37, 46-53,59-68, 80-92, 98-104, 118-143, 150-160, B: 5 191-350 26, 204hypothetical 165-185, 187-200, 204-211, 224-236, 241-246, 252-258,secreted protein 271-280, 288-294, 311-320, 335-341 HP0239 glutamyl-tRNA4-16, 37-59, 64-70, 79-87, 93-102, 107-127, 143-165, H: 2 235-250 27,205 reductase (hemA) 172-188, 197-204, 207-218, 221-227, 242-248,258-277, 289-296, 298-316, 332-338, 344-365, 367-373, 375-382, 400-408,415-425, 438-446 HP0258 conserved 4-37, 39-66, 84-98, 101-127, 140-149,157-163, 166-172, H: 1, L: 1 183-204 28, 206 hypothetical 175-182,184-193, 203-208, 215-232, 234-247, 250-299, integral membrane 303-345protein HP0266 dihydroorotase 10-20, 41-61, 73-87, 112-141, 176-192,194-201, 205-222, M: 21 304-328 29, 207 (pyrC) 230-237, 257-264,276-282, 284-310, 312-318 330-337, 349-357 HP0279 lipopolysaccharide4-31, 42-103, 105-113, 121-153, 160-181, 188-196, 210-226, G: 1, L: 6304-318 30, 208 heptosyltransferase- 231-264, 272-287, 297-304, 328-3361 (rfaC) HP0289 toxin-like outer 21-43, 46-52, 54-70, 72-79, 94-107,133-141, 160-166, B: 1, E: 2, 511-523, 31, 209 membrane protein 217-253,311-317, 359-365, 374-381, 390-395, 434-440, H: 2, I: 3, L: 7 612-630,488-494, 497-502, 511-522, 554-563, 565-574, 577-585, 1790-1803 591-598,601-606, 617-625, 633-643, 658-664, 676-682, 694-702, 710-719, 754-760,782-788, 802-808, 916-921, 942-948, 955-964, 973-979, 992-998,1006-1011, 1016-1023, 1030-1038, 1046-1053, 1059-1066, 1088-1098,1119-1126, 1129-1135, 1156-1171, 1173-1181, 1202-1210, 1255-1261,1268-1280, 1295-1310, 1312-1320, 1375-1381, 1406-1417, 1450-1471,1478-1492, 1498-1506, 1569-1578, 1603-1608, 1611-1624, 1648-1655,1663-1670, 1680-1698, 1702-1707, 1713-1719, 1737-1742, 1747-1753,1762-1769, 1771-1785, 1790-1804, 1811-1818, 1830-1836, 1838-1852,1874-1886, 1893-1899, 1902-1909, 1942-1948, 1952-1962, 1980-1986,2001-2017, 2020-2028, 2042-2050, 2052-2068, 2074-2079, 2083-2095,2107-2113, 2147-2155, 2177-2194, 2203-2211, 2236-2241, 2251-2258,2267-2274, 2285-2292, 2314-2328, 2330-2340, 2358-2365, 2390-2401,2408-2418, 2432-2453, 2463-2476, 2486-2507, 2528-2537, 2540-2548,2552-2558, 2568-2576, 2596-2601, 2610-2622, 2629-2638, 2653-2669,2718-2727, 2749-2767, 2777-2784, 2789-2795, 2806-2815, 2817-2824,2835-2843, 2847-2854, 2860-2881 HP0292 hypothetical protein 4-54, 61-68,72-82, 86-93, 100-108, 115-130, 147-154, E: 14 96-109 32, 210 187-194,196-207, 224-229, 236-251, 275-287 HP0295 flagellin B homolog 31-39,62-69, 91-101, 158-172, 175-180, 186-193, 201-208, A: 1, B: 2, 366-50333, 211 (fla) 210-223, 243-250, 273-286, 293-299, 319-325, 343-354, C: 3356-365, 368-384, 414-435, 471-491, 512-518, 550-556, 567-581, 584-589,633-639, 680-692, 697-708, 716-721, 747-754, 779-786, 810-816 HP0349 CTPsynthetase 5-20, 22-48, 57-65, 96-101, 111-122, 130-145, 154-164, M: 2289-305 34, 212 (pyrG) 170-181, 193-199, 201-216, 224-241, 244-262,281-323, 342-351, 359-367, 369-396, 406-416, 424-433, 450-456, 485-491,493-499, 501-515, 517-535 HP0351 flagellar basal-body 4-17, 22-44,53-60, 66-83, 87-94, 101-106, 110-116, 131-137, G: 9, I: 1 106-126 35,213 M-ring protein (fliF) 148-183, 189-207, 209-215, 233-242, 251-262,264-272, 290-296, 308-327, 359-373, 375-380, 397-405, 415-420, 426-433,444-475, 478-484, 529-536, 548-558 HP0380 glutamate 4-38, 42-50, 58-64,72-81, 92-118, 140-146, 157-165, H: 6, I: 1, L: 4 109-129, 36, 214dehydrogenase 172-192, 198-204, 208-216, 227-234, 238-258, 271-278,370-380 (gdhA) 288-293, 311-322, 327-346, 357-370, 375-383, 395-409,411-417, 425-432, 436-445 HP0392 histidine kinase 23-30, 36-49, 52-64,86-94, 97-104, 121-129, 257-272, B: 1, F: 2 54-72 37, 215 (cheA)279-286, 288-294, 307-327, 334-340, 369-375, 377-386, 406-412, 418-423,430-438, 441-447, 459-465, 469-476, 482-488, 510-546, 550-580, 584-622,638-645, 653-659, 675-683, 692-705, 723-731, 752-761, 788-795 HP04013-phosphoshikimate 11-33, 36-46, 88-104, 116-126, 134-170, 189-195,199-217, E: 1, M: 12 259-273 38, 216 1- 225-250, 255-261, 266-273,280-291, 296-313, 334-341, carboxyvinyltransferase 343-349, 354-360,362-369, 373-380, 387-401, 406-420 (aroA) HP0406 hypothetical protein9-14, 28-44, 57-64, 72-79, 86-93, 104-111, 116-126, 142-150, M: 2 61-8639, 217 159-164 HP0409 GMP synthase 10-17, 26-33, 43-61, 69-95, 101-107,109-125, 129-135, B: 1, F: 1 60-74 40, 218 (guaA) 137-144, 147-153,158-169, 177-187, 209-219, 221-232, 235-247, 261-268, 271-282, 296-302,306-347, 355-362, 364-379, 386-399, 409-418, 424-442, 451-460, 467-479,490-498 HP0413 transposase-like 8-14, 20-31, 65-84, 94-99, 154-179,193-207, 238-253 E: 9, F: 1, G: 1 96-118 41, 219 protein, PS3IS HP0459virB4 homolog 4-24, 30-44, 47-62, 84-93, 108-116, 124-133, 136-141, M: 6167-189 42, 220 (virB4) 201-209, 217-223, 228-235, 238-245, 247-270,275-285, 290-314, 328-338, 342-349, 353-365, 375-383, 386-392, 394-402,417-427, 443-459, 465-481, 492-514, 516-524, 550-566, 602-617, 630-639,666-676, 687-693, 719-730, 747-753, 783-790, 799-816, 824-831, 837-842HP0480 GTP-binding 6-15, 18-28, 58-66, 84-101, 106-129, 136-151,154-165, H: 1 362-375 43, 221 protein, fusA- 182-203, 205-211, 214-220,222-228, 233-240, 251-260, homolog (yihK) 270-277, 284-291, 306-315,322-328, 363-369, 378-388, 392-405, 443-452, 495-501, 512-523, 574-583HP0485 catalase-like protein 5-25, 27-34, 47-59, 64-70, 76-86, 145-158,166-183, 189-202, A: 1, B: 2, 1-102 44, 222 217-231, 235-242, 260-270,278-309 C: 3, K: 2 HP0508 hypothetical protein 4-19, 24-76, 78-83,90-99, 102-109, 114-122, 137-147, A: 1 226-325 45, 223 154-174, 177-188,203-212, 217-223, 227-239 HP0519 conserved 7-37, 71-90, 94-109, 117-128,141-153, 179-192, 199-206, B: 2, H: 1 40-51 46, 224 hypothetical protein225-231, 237-243, 258-264 HP0525 virB11 homolog 13-19, 25-30, 46-59,75-91, 101-107, 114-124, 129-135, D: 5 282-300 47, 225 137-145, 160-167,171-179, 187-194, 209-215, 217-222, 229-239, 243-249, 257-265, 269-275,299-308, 310-327 HP0527 cag pathogenicity 86-100, 216-230, 342-369,382-388, 424-430, 438-445, A: 316, B: 366-781, 48, 226 island protein(cag7- 452-458, 488-494, 501-518, 554-560, 568-574, 584-592, 88, C: 61,F: 782-1518, cagY) 603-609, 611-629, 639-645, 652-661, 669-699, 708-714,12, G: 1, H: 1731-1747 726-738, 747-753, 763-775, 785-791, 794-807,815-824, 1, L: 7, J: 826-845, 854-860, 863-868, 870-883, 892-898,901-906, 274, K: 93, 909-921, 930-937, 946-959, 968-974, 977-990,998-1007, L: 14, M: 12 1009-1027, 1037-1043, 1046-1051, 1053-1066,1075-1081, 1084-1089, 1092-1103, 1113-1119, 1122-1135, 1143-1152,1154-1172, 1182-1188, 1191-1196, 1200-1210, 1220-1226, 1229-1235,1237-1249, 1259-1265, 1268-1281, 1289-1298, 1305-1318, 1328-1334,1337-1343, 1345-1357, 1367-1373, 1390-1396, 1405-1411, 1418-1423,1426-1435, 1445-1455, 1474-1483, 1493-1500, 1505-1512, 1517-1524,1538-1544, 1568-1578, 1595-1601, 1674-1682, 1687-1720, 1728-1736,1738-1744, 1754-1761, 1764-1774, 1798-1824, 1836-1842, 1886-1893,1895-1903 HP0540 cag pathogenicity 4-17, 20-39, 46-55, 60-66, 102-110,114-122, 125-131, A: 2, B: 6, 169-381 49, 227 island protein 161-167,172-178, 185-190, 195-202, 218-232, 236-252, C: 2, I: 1 (cag19) 264-291,293-302, 309-315, 324-339 HP0541 cag pathogenicity 5-10, 13-40, 42-53,69-75, 83-89, 120-135, 150-161, 174-190, B: 2, G: 1 30-200 50, 228island protein 203-225, 229-247, 257-287, 318-348 (cag20) HP0542 cagpathogenicity 7-19, 43-53, 64-72, 124-139 B: 2, L: 18, 52-84, 51, 229island protein M: 2 120-131 (cag21-cagG) HP0544 cag pathogenicity 12-19,39-48, 58-100, 117-123, 154-162, 164-187, 189-195, H: 1 561-575 52, 230island protein 202-216, 218-235, 241-246, 262-278, 315-328, 333-347,(cag23-cagE) 354-366, 372-379, 391-405, 422-429, 431-442, 444-450,458-466, 478-485, 494-501, 504-510, 520-535, 573-580, 589-598, 615-625,666-676, 686-698, 722-729, 737-746, 756-767, 787-796, 805-816, 824-829,833-848, 856-864, 866-876, 879-886, 898-904, 918-924, 927-934, 941-960,967-978 HP0545 cag pathogenicity 11-29, 49-55, 70-77, 84-100, 102-112,148-155, 160-177, A: 1, B: 3, I: 1-104 53, 231 island protein 181-204 2,K: 1 (cag24) HP547 cag pathogenicity 27-44, 64-71, 122-133, 151-156,164-178, 214-220, 226-232, A: 72, B: 65, 110-357, 54, 232 island protein235-244, 253-262, 282-288, 294-310, 317-325, 350-356, C: 175, D:358-501, (cag26-cagA) 362-368, 376-383, 438-443, 449-454, 459-464,492-498, 74, F: 51, G: 502-1161 500-511, 529-535, 538-546, 567-573,597-603, 660-665, 7, H: 10, I: 674-679, 724-734, 763-769, 773-784,791-801, 807-815, 108, J: 9, K: 821-826, 840-848, 863-868, 897-902,908-928, 932-953, 23, L: 33, 956-975, 980-987, 990-996, 1012-1018,1042-1063, M: 129 1095-1116, 1149-1157, 1160-1167 HP0563 hypotheticalprotein 4-21, 64-71, 73-84, 128-138, 144-162, 203-217, 240-263, L: 134330-345 55, 233 288-298, 300-308, 310-317, 325-351, 369-380, 391-411HP0604 uroporphyrinogen 5-11, 25-31, 39-48, 51-79, 89-98, 100-122,135-148, 166-201, A: 2, B: 1, 31-45 56, 234 decarboxylase 203-227,230-250, 254-260, 266-272, 274-282, 299-305, F: 1, K: 2 (hemE) 328-337HP0607 acriflavine 12-23, 29-48, 51-60, 66-72, 75-81, 83-93, 103-115,133-148, F: 2, G: 1, I: 915-940 57, 235 resistance protein 168-174,195-204, 222-229, 231-240, 242-251, 270-280, 1, L: 2 (acrB) 286-305,322-344, 349-360, 364-370, 378-400, 421-441, 448-484, 486-493, 495-501,504-534, 547-561, 567-590, 597-607, 621-635, 643-649, 658-685, 688-694,702-711, 717-731, 737-742, 759-765, 767-772, 776-786, 803-809, 815-825,854-908, 910-919, 923-930, 942-948, 961-975, 994-1014 HP0630 modulatorof drug 4-9, 32-47, 51-61, 75-96, 139-191 A: 1, F: 1, 1-124 58, 236activity (mda66) H: 2, I: 1 HP0635 hypothetical protein 4-13, 17-38,43-49, 55-76, 88-95, 110-121, 128-146, 151-157, F: 2, H: 1 191-203 59,237 162-214, 222-240, 243-249, 251-273, 275-281, 292-298, 300-309,312-320, 322-331, 355-369, 376-408, 446-460, 471-482, 485-509 HP0655protective surface 4-21, 72-82, 89-103, 106-115, 118-124, 140-146,174-184, G: 1, H: 2 33-51, 60, 238 antigen D15 191-200, 204-213,218-224, 261-266, 282-293, 299-309, 818-835 311-340, 342-358, 362-372,381-389, 391-402, 413-421, 438-447, 457-464, 470-478, 501-507, 545-560,578-624, 631-641, 658-670, 680-689, 717-738, 753-759, 795-805, 816-822,830-838, 842-848, 869-881, 892-898 HP0659 hypothetical protein 4-21,79-85, 156-177, 183-188, 206-214, 243-249, 261-269, A: 4, B: 1, 37-97,61, 239 287-292, 315-322, 334-345, 360-366, 374-390, 402-411 K: 2260-399 HP0683 UDP-N- 4-9, 19-54, 58-78, 97-104, 111-120, 126-134,137-145, G: 8 240-249 62, 240 acetylglucosamine 163-173, 178-188,193-203, 211-224, 246-286, 288-324, pyrophosphorylase 337-346, 355-362,374-390, 392-398, 409-417 (glmU) HP0687 iron(II) transport 5-12, 14-31,35-41, 43-61, 82-92, 97-105, 134-145, 155-166, F: 1, L: 3 144-154 63,241 protein (feoB) 184-203, 215-223, 225-251, 272-279, 281-306, 310-345,358-418, 435-473, 482-490, 525-532, 538-547, 549-563, 578-604, 613-639HP0696 N- 53-59, 64-72, 74-100, 133-152, 154-172, 176-181, 207-214, A:5, B: 6, 544-685 64, 242 methylhydantoinase 225-238, 275-297, 304-310,331-340, 362-367, 384-395, C: 13, E: 2, 403-410, 437-443, 448-456,482-490, 579-597, 602-610, F: 1, G: 1, L: 1 625-630, 633-651, 699-707,709-715, 734-743, 750-762 HP0701 DNA gyrase, sub A 12-18, 22-40, 45-83,89-97, 103-109, 147-153, 159-173, G: 2, H: 2, I: 202-218, 65, 243 (gyrA)195-204, 210-219, 243-253, 259-265, 273-282, 303-309, 3, L: 1 282-299,315-325, 332-340, 346-358, 362-367, 377-390, 393-402, 339-350, 418-426,447-455, 467-480, 505-512, 514-525, 548-561, 617-628 566-576, 584-596,619-626, 638-645, 649-659, 661-680, 699-708, 714-720, 753-759, 766-772,775-781, 801-808 HP0706 outer membrane 5-33, 52-62, 87-101, 111-135,137-143, 145-152, 190-202, J: 1, K: 1, L: 151-215 66, 244 protein(omp15) 209-221, 233-245, 253-270 37 HP0714 RNA polymerase 19-29, 32-39,42-48, 75-94, 124-135, 137-145, 152-160, F: 7 48-64 67, 245 sigma-54factor 176-182, 193-203, 215-236, 266-273, 275-291, 297-306, (rpoN)311-319, 322-342, 348-360, 369-378, 394-401 HP0717 DNA polymerase III4-11, 13-33, 36-43, 53-63, 65-80, 112-129, 134-141, 143-155, A: 2, B: 3,390-516 68, 246 gamma and tau 157-168, 178-188, 191-199, 201-207,215-229, 242-255, M: 2 subunits (dnaX) 263-270, 283-315, 320-329,333-338, 340-349, 412-426, 465-478, 485-490, 498-512, 540-554 HP0723L-asparaginase II 4-18, 23-32, 41-47, 54-70, 88-99, 104-111, 118-138,143-148, B: 1, F: 1, 1-90 69, 247 (ansB) 150-162, 168-175, 181-188,203-211, 214-220, 227-245, H: 6, K: 2 251-268, 275-281, 287-296, 323-333HP0727 transcriptional 8-34, 38-49, 72-83, 85-91, 94-104, 112-125,134-142, B: 2, G: 2, I: 2 141-159 70, 248 regulator, putative 148-168,181-189, 191-198, 202-214, 222-233, 242-254, 256-262, 273-278, 287-294,314-325 HP0752 flagellar hook- 4-24, 30-36, 47-75, 82-105, 124-134,151-157, 192-202, A: 7, B: 4, 64-84, 71, 249 associated protein 2208-214, 219-226, 234-247, 285-290, 318-324, 332-340, C: 2, G: 1,93-180, (fliD) 343-349, 380-386, 453-462, 472-478, 484-501, 531-540, H:1, I: 3, J: 1, 181-446 550-557, 604-612, 620-625, 642-648, 652-671 K: 2HP0760 conserved 12-18, 24-32, 68-75, 77-83, 96-101, 109-116, 129-136,F: 3, G: 1 364-374 72, 250 hypothetical protein 152-164, 175-184,190-199, 206-215, 224-233, 241-250, 258-264, 273-292, 302-312, 319-331,334-346, 348-368, 387-395, 408-416, 420-429, 437-452 HP0836 hypotheticalprotein 11-28, 36-52, 60-67, 74-79, 108-116 B: 1, L: 4 61-76 73, 251HP0850 type I restriction 20-27, 38-49, 69-74, 84-107, 138-145, 161-168,179-195, H: 2 146-160 74, 252 enzyme M protein 210-226, 228-252,267-281, 283-296, 305-311, 333-340, (hsdM) 342-356, 361-372, 380-399,401-414, 458-466, 475-481, 492-507, 515-520 HP0853 ABC transporter,43-61, 68-74, 76-90, 120-128, 130-149, 156-161, 164-182, M: 15 261-28575, 253 ATP-binding 206-234, 242-252, 269-274, 291-304, 332-345,349-355, protein (yheS) 360-371, 374-388, 434-440, 447-453, 459-465,469-496, 504-522 HP0863 hypothetical protein 4-17, 24-30, 37-49, 87-98,118-124, 126-136, 144-171, E: 1, M: 11 401-427 76, 254 176-188, 206-214,216-228, 233-240, 246-252, 262-271, 277-297, 307-330, 333-342, 346-352,355-361, 368-386, 391-400, 413-420, 474-480 HP0874 hypothetical protein15-26, 31-46, 51-72, 80-93, 96-109, 131-137, 150-158, F: 2 173-190 77,255 179-185, 189-209, 211-219, 221-234, 241-247, 255-262, 265-271,283-288 HP0875 catalase 28-37, 39-45, 51-58, 77-84, 89-97, 132-148,171-180, K: 6 425-497 78, 256 199-205, 212-218, 220-226, 257-265,273-300, 307-327, 334-340, 344-365, 385-390, 402-408, 426-436, 450-468,476-485 HP0876 iron-regulated outer 4-25, 70-76, 80-88, 90-100, 120-128,162-169, 183-203, B: 1, E: 1, 497-509 79, 257 membrane protein 261-277,279-289, 291-297, 302-308, 321-327, 339-353, G: 1, I: 1 (frpB) 358-377,392-401, 404-410, 414-422, 443-450, 456-461, 470-488, 490-497, 510-535,570-611, 618-630, 639-647, 649-660, 668-690, 702-716, 718-724, 737-747,750-764 HP0887 vacuolating 12-48, 50-64, 99-108, 216-223, 235-241,244-254, 262-274, A: 1, B: 5, 76-100, 80, 258 cytotoxin 287-293,310-316, 320-326, 361-366, 377-383, 390-395, C: 2, D: 3, 270-284,408-414, 418-425, 438-444, 462-469, 494-505, 524-530, E: 5, G: 2,309-438, 536-547, 551-566, 592-598, 601-613, 678-685, 687-695, H: 3, I:2, K: 493-505, 709-717, 727-737, 751-757, 760-765, 772-778, 782-788, 9,L: 29, M: 7 786-942, 801-807, 822-830, 859-868, 870-878, 884-890,898-903, 947-967 909-919, 953-969, 973-980, 990-1000, 1002-1019,1041-1047, 1059-1065, 1090-1095, 1116-1127, 1130-1139, 1143-1149,1151-1168, 1178-1183, 1188-1195, 1197-1209, 1213-1220, 1226-1234,1236-1247, 1255-1274, 1276-1282 HP0891 conserved 4-9, 24-34, 46-95,97-109, 119-130 F: 2 138-156 81, 259 hypothetical protein HP0910 adeninespecific 9-26, 28-35, 43-53, 55-68, 83-92, 99-105, 110-135, 139-149, E:33, H: 1 92-107, 82, 260 DNA 157-162, 164-170, 173-183, 193-208,210-230, 239-245, 154-173 methyltransferase 253-259, 263-271, 293-305,310-320, 322-331, 336-343, (HINDIIM) 351-364, 367-376 HP0913 outermembrane 19-39, 52-62, 108-117, 145-152, 160-168, 194-203, 229-240, C:1, I: 1 247-260 83, 261 protein (omp21) 252-268, 280-287, 308-316,333-339, 383-390, 403-412, 414-424, 438-445, 464-472, 479-484, 489-505,510-526 HP0922 toxin-like outer 5-17, 25-52, 60-77, 105-113, 118-125,162-167, 228-234, A: 1, B: 3, 394-549 84, 262 membrane protein 272-279,328-334, 341-357, 381-395, 400-406, 512-518, D: 1, F: 4, 557-569,586-592, 645-651, 690-695, 701-709, 720-726, G: 2 733-743, 751-758,781-786, 879-886, 929-934, 939-944, 952-960, 965-975, 994-1001,1039-1045, 1102-1109, 1164-1181, 1198-1206, 1223-1229, 1253-1259,1283-1292, 1312-1317, 1339-1349, 1360-1370, 1389-1398, 1400-1412,1452-1465, 1470-1484, 1490-1497, 1519-1525, 1554-1564, 1578-1591,1623-1636, 1638-1646, 1669-1679, 1685-1697, 1704-1711, 1713-1720,1730-1736, 1738-1749, 1756-1764, 1778-1786, 1796-1803, 1817-1826,1849-1866, 1975-1993, 2017-2032, 2044-2053, 2070-2086, 2091-2109,2116-2127, 2156-2167, 2182-2188, 2197-2202, 2244-2252, 2281-2287,2290-2307, 2350-2361, 2383-2404, 2425-2433, 2445-2455, 2495-2505 HP0925recombinational 9-24, 31-53, 57-67, 69-79, 84-114, 133-141, 144-172, E:1, G: 1 13-46 85, 263 DNA repair protein 178-186 (recR) HP0953hypothetical protein 4-25, 27-35, 43-52, 59-70, 79-91, 115-130, 136-152,154-163, J: 3 1-58 86, 264 170-179 HP0973 hypothetical protein 4-30,49-55, 71-80, 96-105, 111-126, 139-146, 149-162, B: 3, K: 2 250-351 87,265 239-245, 279-285, 290-296, 300-307, 331-337, 343-350 HP0977conserved 9-27, 34-41, 43-51, 92-111, 114-120, 123-131, 139-150, A: 2214-398 88, 266 hypothetical 156-171, 176-186, 188-204, 229-241,252-258, 266-279, secreted protein 288-297, 319-334, 338-348, 373-379,389-398, 431-439, 479-484 HP1019 serine protease 4-15, 18-27, 47-52,68-83, 91-97, 104-110, 115-121, 139-147, A: 3, H: 1, 232-386 89, 267(htrA) 157-164, 198-206, 227-236, 241-254, 264-273, 278-289, I: 1311-320, 353-361, 372-383, 405-420, 426-434 HP1024 co-chaperone- 4-10,24-34, 91-97, 129-141, 156-163, 184-190, 205-219, E: 1, G: 1, 93-116 90,268 curved DNA 229-235, 256-273, 278-285 L: 24 binding protein A (CbpA)HP1052 UDP-3-0-acyl N- 7-29, 35-54, 71-83, 85-91, 104-111, 122-134,138-144, E: 1, F: 5, G: 48-66 91, 269 acetylglcosamine 146-154, 158-174,177-183, 186-201, 207-215, 223-235, 14 deacetylase (envA) 240-247,262-273, 275-283, 287-292 HP1090 cell division protein 7-27, 31-47,49-70, 75-102, 110-149, 157-171, 217-223, D: 2, F: 1, 475-490 92, 270(ftsK) 235-251, 294-302, 358-364, 367-375, 387-393, 395-412, G: 1, H: 1423-430, 441-451, 456-470, 472-486, 488-495, 499-509, 515-529, 536-549,556-570, 574-603, 607-615, 625-633, 642-658, 670-676, 683-702, 708-716,720-726, 747-756, 763-784, 803-812, 815-826 HP1098 conserved 7-22,30-38, 53-59, 64-75, 83-95, 97-112, 120-131, 133-142, A: 4, B: 1, 9-156,93, 271 hypothetical 145-151, 154-166, 172-180, 189-203, 227-238,277-287 F: 1, I: 2, K: 1 174-287 secreted protein HP1116 hypotheticalprotein 13-23, 25-32, 111-117, 150-164, 185-193, 207-212, 216-224, I: 1,K: 2, L: 66-86 94, 272 230-236, 263-272, 304-311, 342-348, 374-385,391-407, 31 444-458, 480-487, 489-499, 523-542, 544-558, 572-579,620-640, 686-696, 703-710, 742-755, 765-772, 817-822, 830-837, 865-872,931-937 HP1117 conserved 4-27, 49-56, 62-70, 86-92, 121-127, 151-163,170-182, A: 1, F: 4 234-254 95, 273 hypothetical 195-202, 212-226,237-243 secreted protein HP1119 flagellar hook- 4-10, 13-24, 39-51,62-78, 92-104, 107-117, 134-141, A: 7, B: 1, 252-483 96, 274 associatedprotein 1 156-161, 166-181, 210-216, 222-229, 256-266, 273-280, D: 1, F:3, J: 1 (HAP1) (flgK) 297-304, 313-330, 336-349, 371-376, 433-439,443-448, 488-493, 506-515, 527-534, 560-572, 575-583, 587-593 HP1126colicin tolerance-like 4-15, 21-38, 45-56, 81-95, 102-108, 118-130,133-147, A: 3, B: 2, J: 1 24-146 97, 275 protein (tolB) 152-162,166-171, 199-204, 211-218, 230-240, 253-261, 274-283, 288-294, 312-317,325-336, 344-357, 391-414 HP1152 signal recognition 26-31, 38-56, 65-82,90-101, 112-119, 123-153, 175-188, D: 1, F: 4, 290-306 98, 276 particleprotein (ffh) 197-216, 234-242, 249-265, 273-286, 290-305, 327-335, G:10 338-346, 361-372, 394-404 HP1153 valyl-tRNA 17-26, 43-48, 50-73,81-93, 95-107, 139-146, 158-168, F: 4, G: 3, L: 3 820-851 99, 277synthetase (valS) 171-176, 190-196, 202-212, 216-223, 243-266, 274-282,308-313, 324-330, 344-378, 380-387, 403-422, 427-443, 448-455, 457-465,491-515, 517-528, 553-567, 589-599, 610-617, 642-648, 670-697, 709-717,726-743, 745-759, 769-803, 807-823, 840-849 HP1186 carbonic anhydrase4-18, 39-48, 53-63, 66-90, 102-117, 125-134, 137-145, D: 5, H: 1 26-40,100, 278 156-162, 169-197 56-80 HP1198 DNA-directed RNA 21-33, 36-42,49-60, 68-76, 91-105, 123-130, 141-161, A: 26, B: 14, 213-344, 101, 279polymerase, beta 169-178, 185-190, 192-199, 205-214, 223-233, 239-247,C: 25, D: 3, 954-1080, subunit (rpoB) 260-269, 284-293, 300-314,324-352, 357-364, 373-382, F: 1, G: 3, I: 2524-2733 389-403, 420-432,438-446, 466-471, 477-484, 503-509, 3, J: 31, K: 2 549-556, 558-576,600-623, 625-635, 654-661, 663-669, 671-687, 702-716, 735-741, 744-750,757-766, 776-786, 807-815, 824-832, 854-860, 863-897, 909-915, 920-946,952-959, 982-997, 1024-1038, 1049-1055, 1071-1085, 1104-1113, 1121-1132,1138-1150, 1187-1196, 1212-1221, 1227-1236, 1257-1262, 1264-1278,1282-1294, 1307-1318, 1353-1370, 1382-1388, 1396-1409, 1434-1440,1446-1454, 1465-1478, 1485-1513, 1516-1529, 1540-1545, 1563-1568,1575-1593, 1607-1616, 1628-1645, 1648-1661, 1676-1682, 1689-1697,1713-1719, 1739-1749, 1753-1758, 1763-1774, 1797-1803, 1807-1846,1855-1874, 1877-1891, 1893-1907, 1912-1925, 1931-1943, 1955-1965,1976-1990, 2032-2043, 2045-2051, 2099-2105, 2131-2138, 2161-2179,2188-2199, 2205-2216, 2219-2227, 2235-2245, 2247-2267, 2277-2288,2294-2304, 2314-2326, 2346-2358, 2365-2377, 2383-2402, 2407-2423,2437-2450, 2454-2473, 2489-2497, 2525-2531, 2557-2570, 2580-2587,2589-2599, 2621-2641, 2647-2653, 2661-2677, 2685-2690, 2697-2717,2722-2733, 2739-2777, 2786-2793, 2801-2808, 2811-2822, 2825-2835,2838-2845, 2859-2871, 2877-2883 HP1205 translation 10-16, 18-23, 28-41,63-69, 77-91, 101-109, 118-136, A: 4, B: 1 178-328 102, 280 elongationfactor 146-153, 155-162, 168-179, 192-207, 217-226, 229-235, EF-Tu(tufB) 239-254, 279-286, 294-307, 313-319, 334-341, 344-353, 363-377,390-396 HP1229 aspartokinase (lysC) 18-42, 68-84, 89-95, 100-105,107-115, 125-135, 154-177, I: 1, K: 1, L: 3 85-97 103, 281 189-195,205-228, 236-243, 252-259, 279-300, 309-316, 323-331, 340-351, 353-364,377-402 HP1243 outer membrane 4-18, 26-32, 66-76, 100-126, 151-159,178-186, 188-194, B: 3, F: 1, G: 21-200, 104, 282 protein (omp28)200-210, 241-248, 253-259, 262-279, 284-291, 307-313, 1, H: 1 468-480315-322, 327-337, 376-386, 399-407, 432-441, 467-473, 487-497, 499-505,543-549, 560-568, 585-593, 598-604, 608-614, 630-642, 647-653, 690-703,717-730 HP1254 biotin synthesis 17-49, 52-58, 62-73, 78-97, 100-117,122-172, 185-190, H: 2, I: 2 33-42 105, 283 protein (bioC) 193-217,225-236 HP1265 hypothetical protein 7-39, 50-58, 73-89, 96-107, 109-120,126-142, 152-170, F: 2, G: 6, I: 1 158-169 106, 284 178-202, 205-211,224-244, 249-259, 261-270, 300-310, 312-325 HP1282 anthranilate 4-31,40-64, 71-82, 85-92, 102-124, 126-139, 147-152, E: 20, G: 1, 201-221107, 285 synthase component 159-173, 176-188, 195-207, 210-216, 234-241,249-256, I: 1 I (trpE) 258-276, 279-293, 296-302, 310-315, 349-356,363-378, 380-403, 411-426, 435-441, 448-459, 463-476, 488-494 HP1329cation efflux system 5-13, 15-74, 87-104, 107-120, 123-129, 136-145,150-191, B: 2, F: 1, J: 236-259 108, 286 protein (czcA) 193-206,227-248, 250-264, 278-302, 304-323, 332-378, 1, L: 2 384-407, 409-419,425-457, 462-471, 474-497, 511-545, 555-564, 571-578, 585-598, 640-647,669-675, 682-691, 693-705, 729-743, 752-761, 772-780, 786-804, 808-818,822-846, 858-880, 884-900, 910-939, 941-947, 962-971, 973-988, 998-1003,1007-1027 HP1339 biopolymer 4-19, 27-68, 81-111, 121-160 F: 5, I: 460-79 109, 287 transport protein (exbB) HP1341 siderophore- 4-37, 40-46,52-57, 199-205, 222-229, 236-244, 250-267, A: 20, B: 23, 27-197 110, 288mediated iron 269-282 C: 30, E: 31, transport protein F: 5, G: 3, H: 6,I: 1, J: 2, K: 5 HP1342 outer membrane 4-16, 24-30, 32-38, 63-75, 86-92,98-111, 113-126, A: 1, B: 3, 546-559 111, 289 protein (omp29) 160-165,170-180, 198-204, 227-233, 239-245, 253-273, C: 1, D: 1, 308-314,352-365, 382-387, 395-403, 423-429, E: 1, F: 2, 472-482, 484-493,501-507, 518-526, 536-541, 543-550, H: 7 556-562, 586-600, 626-633,649-661, 680-688 HP1345 phosphoglycerate 16-33, 48-59, 63-71, 77-92,94-109, 117-124, 139-151, D: 20, I: 6 258-272 112, 290 kinase 169-181,184-227, 233-249, 251-261, 263-275, 282-294, 297-321, 326-332, 341-355,383-399 HP1350 protease 11-26, 31-39, 43-52, 55-62, 64-70, 80-94,123-133, 135-141, A: 3, B: 1, 77-226, 113, 291 172-181, 185-206,209-218, 224-230, 238-244, 251-262, C: 1, G: 1, 350-429 264-271,290-301, 306-324, 333-340, 350-357, 367-375, K: 2, M: 12 390-397,434-441, 443-448 HP1374 ATP-dependent 4-13, 22-27, 31-45, 50-59, 72-96,99-114, 131-141, 143-150, G: 9, M: 2 283-305 114, 292 protease ATPase159-176, 180-186, 189-198, 208-214, 234-253, 271-287, subunit (clpX)294-299, 310-366, 382-390, 398-416, 424-443 HP1393 DNA repair protein9-26, 30-53, 62-72, 86-95, 112-122, 136-145, 153-160, E: 1, F: 2, G:213-232 115, 293 (recN) 209-221, 227-237, 241-268, 281-288, 291-298,308-314, 7, I: 1 321-328, 336-346, 351-379, 388-397, 409-416, 423-433,443-481, 511-519 HP1448 ribonuclease P, 12-18, 25-31, 38-50, 59-67,71-82, 96-126 G: 6 76-88 116, 294 protein component (rnpA) HP1453conserved 4-25, 39-44, 64-71, 74-88, 100-113, 128-138, 151-162, B: 1, D:4, 2-100 117, 295 hypothetical protein 164-177, 185-190, 204-213,233-239, 246-254, 281-286, F: 2, J: 1, K: 293-306, 309-318, 333-347,349-359, 385-398, 404-423, 11, L: 3 458-465, 477-484, 490-499, 501-533,554-566, 582-590, 596-616, 624-629, 631-639, 654-680, 694-720, 735-743HP1454 hypothetical protein 4-16, 36-41, 52-75, 98-107, 109-117,122-128, 133-139, B: 1, M: 8 75-92 118, 296 141-155, 159-165, 169-182,187-193, 195-201, 211-224, 230-236, 247-269, 278-290 HP1460 DNApolymerase III 7-21, 25-33, 37-43, 87-94, 103-120, 131-147, 168-174, F:3, G: 2, I: 3 732-748 119, 297 alpha-subunit 197-203, 207-212, 227-237,247-257, 263-271, 279-287, (dnaE) 298-306, 320-325, 332-340, 363-374,379-384, 390-401, 403-414, 428-433, 448-457, 462-475, 483-490, 513-519,525-535, 543-554, 559-566, 571-620, 625-631, 636-642, 659-670, 688-706,708-723, 770-779, 787-793, 796-807, 820-840, 848-854, 863-874, 895-905,912-919, 934-942, 968-975, 983-1000, 1012-1019, 1026-1036, 1050-1060,1064-1070, 1081-1091, 1094-1108, 1112-1118, 1140-1152, 1164-1169,1172-1180, 1187-1192 HP1497 peptidyl-tRNA 23-40, 42-59, 66-73, 78-97,111-128, 130-141, 157-166, E: 6, F: 1, 53-71 120, 298 hydrolase (pth)178-183 H: 2 HP1527 hypothetical protein 4-27, 38-44, 47-57, 59-85,99-106, 114-121, 154-166, B: 3, C: 1, I: 7 86-195 121, 299 181-186,193-198, 238-244, 253-262, 272-278, 287-299, 314-320, 338-350, 358-368,382-388, 407-416, 433-446, 456-461, 463-473 HP1564 outer membrane 5-24,38-59, 64-80, 87-99, 105-126, 134-142, 149-163, A: 2, B: 1, 87-245 122,300 protein 165-179, 181-202, 205-220, 227-233, 243-250, 257-263 C: 3,F: 1, H: 1 HP1565 pencillin-binding 5-32, 47-53, 66-79, 81-97, 115-151,155-174, 183-188, A: 1, F: 2, 347-371, 123, 301 protein 2 (pbp2)196-210, 215-226, 230-238, 253-258, 263-270, 276-282, H: 1, L: 5 375-386295-301, 304-325, 334-344, 360-390, 397-412, 425-432, 434-462, 478-494,508-526, 539-564, 571-579 HP1574 riboflavin synthase 4-15, 36-44, 49-56,60-66, 68-82, 84-103, 109-115, 118-141, H: 1, M: 5 26-39 124, 302 alphasubunit (ribC) 147-154, 160-168, 176-185 ARF0044 Hypothetical 7-13,23-33 H: 1, I: 2 13-21 125, 303 protein ARF0048 Hypothetical none F: 1,G: 1, I: 2-10 126, 304 protein 4, L: 8 ARF0143 Hypothetical 4-9, 12-18,35-42, 49-62 F: 1 6-18 127, 305 protein ARF0184 Hypothetical 19-25 A: 1,I: 6 1-13 128, 306 protein ARF0219 Hypothetical 15-21, 27-45 E: 7 12-25129, 307 protein ARF0308 Hypothetical 14-20 E: 41 1-14 130, 308 proteinARF0349 Hypothetical 4-18 G: 11 13-26 131, 309 protein ARF0387Hypothetical 8-21 G: 12, H: 2, 2-20 132, 310 protein K: 1 ARF0402Hypothetical 4-14 F: 2, G: 10, 4-16 133, 311 protein H: 1, M: 2 ARF0501Hypothetical none M: 2 3-12 134, 312 protein ARF0509 Hypothetical 6-14,6-25, 35-57 G: 5, H: 1, 2-14 135, 313 protein M: 3 ARF0522 Hypothetical6-25, 35-57 F: 1, I: 3, K: 2 17-31 136, 314 protein ARF0578 Hypothetical14-25, 32-46 E: 12, G: 1 5-19 137, 315 protein ARF0629 Hypothetical18-31 G: 1, I: 7 5-16 138, 316 protein ARF0665 Hypothetical 19-24 L: 2,M: 5 4-26 139, 317 protein ARF0693 Hypothetical 13-21, 29-34, 47-58,61-73 G: 1, I: 6 36-47 140, 318 protein ARF0752 Hypothetical 4-15 D: 35-24 141, 319 protein ARF0788 Hypothetical none B: 1, H: 1 6-18 142, 320protein ARF0819 Hypothetical 13-20 F: 14 4-13 143, 321 protein ARF0839Hypothetical none A: 2, C: 1, I: 15-23 144, 322 protein 1, K: 3 ARF0868Hypothetical 4-9 F: 2, G: 1 7-21 145, 323 protein ARF0948 Hypotheticalnone F: 1 1-10 146, 324 protein ARF0969 Hypothetical none B: 1, G: 1,4-14 147, 325 protein M: 2 ARF1100 Hypothetical 4-17, 35-41, 46-89,93-98 A: 1, B: 1, 70-88 148, 326 protein H: 1, I: 2, M: 5 ARF1164Hypothetical none G: 1, H: 3, J: 1-13 149, 327 protein 1, M: 2 ARF1470Hypothetical 4-16, 26-32 G: 1, I: 1, 25-38 150, 328 protein M: 2 ARF1553Hypothetical 8-15, 23-28 B: 2, F: 6, G: 4-17 151, 329 protein 2, H: 1,I: 1, K: 1 CRF0017 Hypothetical 4-12 H: 1, I: 1, 1-15 152, 330 proteinM: 5 CRF0025 Hypothetical 4-29, 31-42, 52-58 E: 1, G: 1, 6-16 153, 331protein L: 3, M: 1 CRF0090 Hypothetical 4-9, 24-32 F: 13, G: 1, 9-19154, 332 protein H: 1, L: 1 CRF0127 Hypothetical 4-12, 18-27 L: 3, M: 105-18 155, 333 protein CRF0169 Hypothetical 4-11, 37-56, 58-92 D: 3 18-29156, 334 protein CRF0190 Hypothetical 8-28 M: 14 20-35 157, 335 proteinCRF0251 Hypothetical none D: 16, E: 2, 4-15 158, 336 protein G: 3CRF0258 Hypothetical 4-23, 27-39, 55-63 A: 1, B: 1, 35-58 159, 337protein C: 1, F: 1, L: 5 CRF0354 Hypothetical 6-26, 28-54 F: 8, H: 128-47 160, 338 protein CRF0388 Hypothetical 4-10, 38-52, 58-82 H: 1, L:3 30-49 161, 339 protein CRF0409 Hypothetical 4-22, 29-35, 44-50, 53-68,70-80 G: 2, H: 1, 20-33 162, 340 protein M: 10 CRF0421 Hypothetical22-28, 30-36 F: 4 18-33 163, 341 protein CRF0480 Hypothetical 4-11,13-21, 25-30 E: 1, L: 8 20-30 164, 342 protein CRF0552 Hypothetical10-22 G: 1, M: 5 10-23 165, 343 protein CRF0563 Hypothetical 4-11 G: 1,L: 1, 9-20 166, 344 protein M: 6 CRF0578 Hypothetical 14-25, 32-46 G: 1,L: 7, 6-19 167, 345 protein M: 12 CRF0626 Hypothetical 5-30 L: 43 14-33168, 346 protein CRF0870 Hypothetical 4-15, 28-35, 46-55, 59-65, 76-84H: 6, I: 1, L: 8 9-24 169, 347 protein CRF0894 Hypothetical 27-33 L: 55-19 170, 348 protein CRF0922 Hypothetical 5-13 E: 11, F: 3, 8-18 171,349 protein G: 1, H: 7, I: 1, L: 4 CRF1012 Hypothetical 9-22, 24-34 L: 321-40 172, 350 protein CRF1100 Hypothetical 4-17, 35-41, 46-89, 93-98 E:1, H: 5, I: 71-89 173, 351 protein 2, L: 4 CRF1301 Hypothetical 4-12,14-24 H: 9, I: 2 2-17 174, 352 protein CRF1354 Hypothetical 9-17 I: 2,M: 16 5-16 175, 353 protein CRF1422 Hypothetical 7-41, 48-58, 63-75,80-89 G: 2, H: 1, 43-53 176, 354 protein L: 62 CRF1489 Hypothetical4-22, 25-30 E: 1, F: 1, 4-14 177, 355 protein H: 5, L: 10 CRF1549Hypothetical 4-55 G: 1, M: 7 18-33 178, 356 protein

TABLE 2 Gene distribution in H. pylori strains. H. pylori antigenicPutative function Gene distribution Amino acid substitutions (in Seq. IDprotein (by homology) (presence in 28 strains) cancer patient isolate)*(DNA, Prot.) HP0009 outer membrane protein (omp1) n.d. n.d. 1, 179HP0010 chaperone and heat shock n.d. n.d. 2, 180 protein (groEL) HP0043mannose-6-phosphate isomerase 28/28 11/247 3, 181 (pmi) HP0063hypothetical protein n.d. n.d. 4, 182 HP0067 urease accessory protein(ureH) 13/28  9/230 5, 183 HP0072 urease beta subunit (urea n.d. n.d. 6,184 amidohydrolase) (ureB) HP0086 Conserved hypothetical protein 28/28 1/221 7, 185 HP0087 hypothetical protein 28/28 15/241 8, 186 HP0088 RNApolymerase sigma-70 factor 28/28  4/226 9, 187 (rpoD) HP0089 pfs protein(pfs) 28/28  5/199 10, 188 HP0115 flagellin B (flaB) 28/28  2/249 11,189 HP0175 cell binding factor 2 n.d. n.d. 12, 190 HP0121phosphoenolpyruvate synthase 28/28  0/240 13, 191 (ppsA) HP0123threonyl-tRNA synthetase (thrS) 28/28  7/238 14, 192 HP0130 hypotheticalprotein 28/28  8/241 15, 193 HP0150 hypothetical protein 28/28  5/11116, 194 HP0183 serine hydroxymethyltransferase 28/28  2/227 17, 195(glyA) HP0192 fumarate reductase, flavoprotein 28/28  3/219 18, 196subunit (frdA) HP0197 S-adenosylmethionine 28/28  1/243 19, 197synthetase 2 (metX) HP0201 fatty acid/phospholipid synthesis 28/28 0/176 20, 198 protein (plsX) HP0202 beta-ketoacyl-acyl carrier protein28/28  7/232 21, 199 synthase III HP0210 chaperone and heat shock n.d.n.d. 22, 200 protein C62.5 (htpG) HP0211 conserved hypothetical secreted28/28  3/201 23, 201 protein HP0228 conserved hypothetical integral28/28  3/248 24, 202 membrane protein HP0229 outer membrane protein(omp6) n.d. n.d. 25, 203 HP0235 conserved hypothetical secreted n.d.n.d. 26, 204 protein HP0239 glutamyl-tRNA reductase  7/28   4/257^(#)27, 205 (hemA) HP0258 conserved hypothetical integral 28/28 14/254 28,206 membrane protein HP0266 dihydroorotase (pyrC) 15/28 14/253 29, 207HP0279 lipopolysaccharide 28/28 15/246 30, 208 heptosyltransferase-1(rfaC) HP0289 toxin-like outer membrane 28/28 12/241 31, 209 proteinHP0292 hypothetical protein 28/28  9/245 32, 210 HP0295 flagellin Bhomolog (fla) 14/28   9/224^(#) 33, 211 HP0349 CTP synthetase (pyrG)28/28  2/249 34, 212 HP0351 flagellar basal-body M-ring 28/28  2/249 35,213 protein (fliF) HP0380 glutamate dehydrogenase 28/28  9/253 36, 214(gdhA) HP0392 histidine kinase (cheA) 27/28 4/259; 5 aa inserted, 4 aa37, 215 deleted HP0401 3-phosphoshikimate 1- 28/28 14/248 38, 216carboxyvinyltransferase (aroA) HP0406 hypothetical protein 28/28  2/14639, 217 HP0409 GMP synthase (guaA) 28/28  9/252 40, 218 HP0413transposase-like protein, PS3IS 10/28   0/268^(#) 41, 219 HP0459 virB4homolog (virB4)  9/28   6/248^(#) 42, 220 HP0480 GTP-binding protein,fusA- n.d. n.d. 43, 221 homolog (yihK) HP0485 catalase-like protein28/28  7/252 44, 222 HP0508 hypothetical protein 28/28  3/260 45, 223HP0519 conserved hypothetical protein 28/28 6/224; 1 aa inserted, 1 aa46, 224 deleted HP0525 vitB11 homolog 24/28  0/257 47, 225 HP0527 cagpathogenicity island protein 22/28  4/214 48, 226 (cag7-cagY) HP0540 cagpathogenicity island protein 25/28  5/251 49, 227 (cag19) HP0541 cagpathogenicity island protein 26/28  3/251 50, 228 (cag20) HP0542 cagpathogenicity island protein 23/28  0/115 51, 229 (cag21-cagG) HP0544cag pathogenicity island protein 11/28 n.d. 52, 230 (cag23-cagE) HP0545cag pathogenicity island protein 25/28  1/153 53, 231 (cag24) HP547 cagpathogenicity island protein 24/28 20/256; 1 aa inserted^(#) 54, 232(cag26-cagA) HP0563 hypothetical protein 28/28  9/192 55, 233 HP0604uroporphyrinogen 28/28  5/260 56, 234 decarboxylase (hemE) HP0607acriflavine resistance protein 28/28  0/254 57, 235 (acrB) HP0630modulator of drug activity 28/28   2/112^(#) 58, 236 (mda66) HP0635hypothetical protein 24/28 13/135 59, 237 HP0655 protective surfaceantigen D15 28/28 n.d. 60, 238 HP0659 hypothetical protein  6/28 14/18761, 239 HP0683 UDP-N-acetylglucosamine 28/28  8/193 62, 240pyrophosphorylase (glmU) HP0687 iron(II) transport protein (feoB) 28/28 2/203 63, 241 HP0696 N-methylhydantoinase 28/28  2/206 64, 242 HP0701DNA gyrase, sub A (gyrA) 28/28  5/224 65, 243 HP0706 outer membraneprotein (omp15) 28/28  2/167 66, 244 HP0714 RNA polymerase sigma-54factor 28/28  8/200 67, 245 (rpoN) HP0717 DNA polymerase III gamma and28/28 15/137; 2 aa inserted^(#) 68, 246 tau subunits (dnaX) HP0723L-asparaginase II (ansB) 28/28 12/220 69, 247 HP0727 transcriptionalregulator, 28/28  4/207 70, 248 putative HP0752 flagellarhook-associated protein 28/28  2/191 71, 249 2 (fliD) HP0760 conservedhypothetical protein 28/28  2/211 72, 250 HP0836 hypothetical protein28/28 1/82 73, 251 HP0850 type I restriction enzyme M 28/28 10/181 74,252 protein (hsdM) HP0853 ABC transporter, ATP-binding 19/28   2/198^(#)75, 253 protein (yheS) HP0863 hypothetical protein 27/28  2/161 76, 254HP0874 hypothetical protein 28/28  3/243 77, 255 HP0875 catalase n.d.n.d. 78, 256 HP0876 iron-regulated outer membrane 28/28  4/193 79, 257protein (frpB) HP0887 vacuolating cytotoxin 28/28   9/228^(#) 80, 258HP0891 conserved hypothetical protein 18/28   2/149^(#) 81, 259 HP0910adenine specific DNA 27/28  3/205 82, 260 methyltransferase (HINDIIM)HP0913 outer membrane protein (omp21) 28/28 12/172; 1 aa deleted 83, 261HP0922 toxin-like outer membrane 28/28 27/198 84, 262 protein HP0925recombinational DNA repair 28/28  1/159 85, 263 protein (recR) HP0953hypothetical protein 28/28  2/164 86, 264 HP0973 hypothetical protein19/28 6/248; 1 aa deleted 87, 265 HP0977 conserved hypothetical secreted28/28  9/238 88, 266 protein HP1019 serine protease (htrA) n.d. n.d. 89,267 HP1024 co-chaperone-curved DNA 25/28  5/167 90, 268 binding proteinA (CbpA) HP1052 UDP-3-0-acyl N- 28/28  5/186 91, 269 acetylglcosaminedeacetylase (envA) HP1090 cell division protein (ftsK) 28/28  2/223 92,270 HP1098 conserved hypothetical secreted n.d. n.d. 93, 271 proteinHP1116 hypothetical protein 28/28 192/283  94, 272 HP1117 conservedhypothetical secreted n.d. n.d. 95, 273 protein HP1119 flagellarhook-associated protein 28/28  7/213 96, 274 1 (HAP1) (flgK) HP1126colicin tolerance-like protein 28/28  4/241 97, 275 (tolB) HP1152 signalrecognition particle n.d. n.d. 98, 276 protein (ffh) HP1153 valyl-tRNAsynthetase (valS) 28/28 14/243 99, 277 HP1186 carbonic anhydrase 100,278 HP1198 DNA-directed RNA polymerase, 28/28  7/232 101, 279 betasubunit (rpoB) HP1205 translation elongation factor EF- n.d. n.d. 102,280 Tu (tufB) HP1229 aspartokinase (lysC) 2728   4/245^(#) 103, 281HP1243 outer membrane protein (omp28) n.d. n.d. 104, 282 HP1254 biotinsynthesis protein (bioC) 28/28  9/169 105, 283 HP1265 hypotheticalprotein 28/28 19/216 106, 284 HP1282 anthranilate synthase component28/28 12/193 107, 285 I (trpE) HP1329 cation efflux system protein 28/28 3/196 108, 286 (czcA) HP1339 biopolymer transport protein 27/28  1/109^(#) 109, 287 (exbB) HP1341 siderophore-mediated iron 28/28 12/179^(#) 110, 288 transport protein HP1342 outer membrane protein(omp29) n.d. n.d. 111, 289 HP1345 phosphoglycerate kinase 28/28  3/220112, 290 HP1350 protease n.d. n.d. 113, 291 HP1374 ATP-dependentprotease ATPase 28/28  0/211 114, 292 subunit (clpX) HP1393 DNA repairprotein (recN) 28/28  4/209 115, 293 HP1448 ribonuclease P, protein19/28   5/124^(#) 116, 294 component (rnpA) HP1453 conservedhypothetical protein 28/28  8/200 117, 295 HP1454 hypothetical proteinn.d. n.d. 118, 296 HP1460 DNA polymerase III alpha- 28/28  2/225 119,297 subunit (dnaE) HP1497 peptidyl-tRNA hydrolase (pth) 28/28  4/155120, 298 HP1527 hypothetical protein 28/28 14/202 121, 299 HP1564 outermembrane protein n.d. n.d. 122, 300 HP1565 penicillin-binding protein 228/28  3/178 123, 301 (pbp2) HP1574 riboflavin synthase alpha 23/28  4/153^(#) 124, 302 subunit (ribC) ARF0044 Hypothetical protein n.d.n.d. 125, 303 ARF0048 Hypothetical protein n.d. n.d. 126, 304 ARF0143Hypothetical protein n.d. n.d. 127, 305 ARF0184 Hypothetical proteinn.d. n.d. 128, 306 ARF0219 Hypothetical protein n.d. n.d. 129, 307ARF0308 Hypothetical protein n.d. n.d. 130, 308 ARF0349 Hypotheticalprotein n.d. n.d. 131, 309 ARF0387 Hypothetical protein n.d. n.d. 132,310 ARF0402 Hypothetical protein n.d. n.d. 133, 311 ARF0501 Hypotheticalprotein n.d. n.d. 134, 312 ARF0509 Hypothetical protein n.d. n.d. 135,313 ARF0522 Hypothetical protein n.d. n.d. 136, 314 ARF0578 Hypotheticalprotein n.d. n.d. 137, 315 ARF0629 Hypothetical protein n.d. n.d. 138,316 ARF0665 Hypothetical protein n.d. n.d. 139, 317 ARF0693 Hypotheticalprotein n.d. n.d. 140, 318 ARF0752 Hypothetical protein n.d. n.d. 141,319 ARF0788 Hypothetical protein n.d. n.d. 142, 320 ARF0819 Hypotheticalprotein n.d. n.d. 143, 321 ARF0839 Hypothetical protein n.d. n.d. 144,322 ARF0868 Hypothetical protein n.d. n.d. 145, 323 ARF0948 Hypotheticalprotein n.d. n.d. 146, 324 ARF0969 Hypothetical protein n.d. n.d. 147,325 ARF1100 Hypothetical protein n.d. n.d. 148, 326 ARF1164 Hypotheticalprotein n.d. n.d. 149, 327 ARF1470 Hypothetical protein n.d. n.d. 150,328 ARF1553 Hypothetical protein n.d. n.d. 151, 329 CRF0017 Hypotheticalprotein n.d. n.d. 152, 330 CRF0025 Hypothetical protein n.d. n.d. 153,331 CRF0090 Hypothetical protein n.d. n.d. 154, 332 CRF0127 Hypotheticalprotein n.d. n.d. 155, 333 CRF0169 Hypothetical protein n.d. n.d. 156,334 CRF0190 Hypothetical protein n.d. n.d. 157, 335 CRF0251 Hypotheticalprotein n.d. n.d. 158, 336 CRF0258 Hypothetical protein n.d. n.d. 159,337 CRF0354 Hypothetical protein n.d. n.d. 160, 338 CRF0388 Hypotheticalprotein n.d. n.d. 161, 339 CRF0409 Hypothetical protein n.d. n.d. 162,340 CRF0421 Hypothetical protein n.d. n.d. 163, 341 CRF0480 Hypotheticalprotein n.d. n.d. 164, 342 CRF0552 Hypothetical protein n.d. n.d. 165,343 CRF0563 Hypothetical protein n.d. n.d. 166, 344 CRF0578 Hypotheticalprotein n.d. n.d. 167, 345 CRF0626 Hypothetical protein n.d. n.d. 168,346 CRF0870 Hypothetical protein n.d. n.d. 169, 347 CRF0894 Hypotheticalprotein n.d. n.d. 170, 348 CRF0922 Hypothetical protein n.d. n.d. 171,349 CRF1012 Hypothetical protein n.d. n.d. 172, 350 CRF1100 Hypotheticalprotein n.d. n.d. 173, 351 CRF1301 Hypothetical protein n.d. n.d. 174,352 CRF1354 Hypothetical protein n.d. n.d. 175, 353 CRF1422 Hypotheticalprotein n.d. n.d. 176, 354 CRF1489 Hypothetical protein n.d. n.d. 177,355 CRF1549 Hypothetical protein n.d. n.d. 178, 356

TABLE 3

1-37. (canceled)
 38. A hyperimmune serum-reactive antigen comprising anamino acid sequence from any of SEQ ID NOs: 179-356 or fragmentsthereof.
 39. The hyperimmune serum-reactive antigen or fragment of claim38, further defined as a peptide comprising an amino acid sequencedescribed in the “predicted immunogenic aa” and/or “location ofidentified immunogenic region” of Table 1 or of a serum reactive epitopeof Table
 3. 40. The hyperimmune serum reactive antigen or fragment ofclaim 38, further defined as comprising an amino acid sequence of aminoacids: 63-91, 95-101, 110-116, 134-148, 150-156, 158-164, 188-193,197-209, 226-241, 247-254, 291-297, 312-319, 338-346, 351-358, 366-378,404-410, 420-438, 448-454, 465-473, 482-488, 490-498, 503-510, 512-519,531-543, 547-554, 568-575, 589-604, 610-631 and 239-308 of SEQ IDNO:179; 16-29, 35-47, 50-68, 70-79, 91-101, 143-149, 158-163, 185-191,196-206, 215-224, 230-237, 244-251, 258-278, 290-311, 319-325, 338-351,365-385, 396-429, 445-454, 458-466, 491-499, 501-521, 17-79 and 218-233of SEQ ID NO:180; 4-10, 16-41, 46-66, 77-84, 91-97, 102-118, 125-144,187-200, 202-214, 245-253, 255-261, 286-295, 300-330, 335-342, 350-361,363-381, 385-392, 396-416, 435-450 and 460-470 of SEQ ID NO:181; 11-19,27-48, 52-59, 77-82, 84-107, 118-125, 127-154, 178-183, 192-209,215-221, 286-295, 302-313, 350-357, 402-415, 417-431, 453-463, 465-493and 313-331 of SEQ ID NO:182; 19-26, 30-43, 47-55, 63-68, 72-80, 97-104,107-119, 129-146, 160-175, 194-216, 231-251, 254-260 and 26-43 of SEQ IDNO:183; 7-13, 29-37, 65-81, 110-120, 123-131, 135-152, 230-249, 254-260,284-290, 292-299, 317-326, 329-336, 403-444, 452-458, 466-477, 490-498,510-519, 541-550, 557-566 and 533-567 of SEQ ID NO:184; 5-47, 71-77,79-86, 89-95, 120-126, 137-144, 176-181, 184-196, 202-208, 211-232,236-282, 301-313, 317-325, 341-347, 353-384, 394-400, 412-433, 436-443and 59-75 of SEQ ID NO:185; 4-18, 22-38, 59-69, 106-112, 116-130,138-149, 156-170, 175-197, 200-214, 216-223, 233-244, 255-261, 266-276,279-286, 325-333, 342-348, 366-399, 402-420, 429-441, 1-104 and 130-147of SEQ ID NO:186; 50-58, 69-95, 97-113, 131-136, 157-163, 170-175,188-212, 220-226, 254-259, 265-277, 283-289, 297-308, 311-318, 347-358,360-369, 378-401, 416-421, 440-450, 454-462, 470-476, 493-502, 506-514,536-567, 585-590, 598-607, 613-618, 653-659 and 35-46 of SEQ ID NO:187;16-29, 32-60, 65-87, 89-123, 128-134, 137-158, 162-173, 178-196,210-216, 218-228 and 206-225 of SEQ ID NO:188; 10-20, 26-35, 51-64,86-91, 94-100, 113-122, 154-160, 185-191, 193-201, 211-217, 225-230,237-246, 251-257, 298-304, 306-312, 316-328, 340-348, 357-389, 391-397,415-421, 449-456, 458-471, 488-495, 502-511, 24-55 and 236-341 of SEQ IDNO:189; 5-22, 41-51, 87-93, 114-122, 127-136, 150-156, 158-166, 223-233,245-263, 291-296, 9-126 and 127-285 of SEQ ID NO:190; 30-43, 46-56,61-70, 72-83, 85-93, 103-113, 119-125, 151-166, 179-191, 212-218,225-231, 236-243, 262-267, 291-307, 331-344, 349-355, 366-372, 380-386,414-422, 428-447, 459-464, 469-478, 507-519, 525-544, 563-569, 576-590,620-626, 633-643, 654-659, 665-671, 684-707, 717-723, 725-733, 747-779,782-801 and 347-361 of SEQ ID NO:191; 4-12, 14-26, 37-80, 107-115,133-139, 144-150, 154-165, 173-180, 191-199, 205-211, 221-231, 237-244,254-284, 307-340, 342-353, 360-368, 370-380, 479-493, 495-503, 509-522,525-536, 539-547, 554-560, 565-573, 578-583, 7-23 and 465-479 of SEQ IDNO:192; 4-17, 47-55, 76-83, 85-100, 104-112, 117-123, 126-135, 142-148,156-167, 174-182, 267-273 and 258-283 of SEQ ID NO:193; 8-32, 36-42,65-88, 102-108, 112-140, 147-163, 170-179, 183-193 and 117-124 of SEQ IDNO:194; 12-18, 45-50, 62-77, 82-95, 99-113, 115-123, 125-147, 155-177,187-209, 211-223, 244-253, 259-270, 278-297, 302-307, 311-318, 329-334,350-356, 359-365, 390-400, 402-413 and 333-350 of SEQ ID NO:195; 4-13,15-27, 30-46, 53-58, 68-74, 82-95, 115-126, 134-139, 148-153, 159-176,182-199, 201-217, 220-225, 227-235, 237-248, 253-266, 300-315, 322-336,390-396, 412-426, 438-445, 448-459, 477-484, 502-508, 515-527, 529-537,553-568, 643-651, 658-667, 690-703 and 376-400 of SEQ ID NO:196; 4-10,24-32, 38-55, 59-67, 70-77, 80-87, 89-97, 123-129, 134-151, 166-172,178-189, 191-216, 218-235, 245-259, 271-315, 326-339, 341-360 and 73-94of SEQ ID NO:197; 13-25, 31-38, 43-57, 79-85, 92-99, 106-112, 117-128,130-139, 146-158, 160-175, 194-204, 211-222, 225-232, 234-242, 263-270,278-292, 299-320, 322-333 and 240-256 of SEQ ID NO:198; 4-17, 55-63,66-101, 109-131, 135-143, 145-151, 155-161, 164-170, 177-185, 192-198,213-218, 223-238, 246-256, 258-268, 273-283, 309-314, 322-328 and195-221 of SEQ ID NO:199; 13-24, 31-39, 41-50, 63-69, 90-96, 104-109,116-141, 148-153, 161-167, 173-178, 190-209, 253-258, 265-272, 279-289,295-312, 317-343, 355-366, 376-389, 400-407, 430-451, 453-464, 466-472,487-493, 499-505, 523-538, 554-559, 568-579, 584-601 and 344-363 of SEQID NO:200; 5-22, 30-36, 53-59, 61-70, 82-92, 99-106, 120-131, 135-148,154-167, 169-183, 187-199, 204-212, 231-247 and 111-249 of SEQ IDNO:201; 17-36, 40-66, 71-144, 148-171, 173-191, 199-214, 220-252,265-272, 278-288, 298-333, 342-385 and 287-307 of SEQ ID NO:202; 4-16,22-28, 30-36, 42-48, 95-116, 154-162, 164-174, 239-252, 258-263,273-285, 306-313, 323-333, 341-357, 363-369, 372-379, 395-401, 430-436,438-453, 464-480, 33-44, 233-258 and 349-369 of SEQ ID NO:203; 4-21,30-37, 46-53, 59-68, 80-92, 98-104, 118-143, 150-160, 165-185, 187-200,204-211, 224-236, 241-246, 252-258, 271-280, 288-294, 311-320, 335-341and 191-350 of SEQ ID NO:204; 4-16, 37-59, 64-70, 79-87, 93-102,107-127, 143-165, 172-188, 197-204, 207-218, 221-227, 242-248, 258-277,289-296, 298-316, 332-338, 344-365, 367-373, 375-382, 400-408, 415-425,438-446 and 235-250 of SEQ ID NO:205; 4-37, 39-66, 84-98, 101-127,140-149, 157-163, 166-172, 175-182, 184-193, 203-208, 215-232, 234-247,250-299, 303-345 and 183-204 of SEQ ID NO:206; 10-20, 41-61, 73-87,112-141, 176-192, 194-201, 205-222, 230-237, 257-264, 276-282, 284-310,312-318, 330-337, 349-357 and 304-328 of SEQ ID NO:207; 4-31, 42-103,105-113, 121-153, 160-181, 188-196, 210-226, 231-264, 272-287, 297-304,328-336 and 304-318 of SEQ ID NO:208; 21-43, 46-52, 54-70, 72-79,94-107, 133-141, 160-166, 217-253, 311-317, 359-365, 374-381, 390-395,434-440, 488-494, 497-502, 511-522, 554-563, 565-574, 577-585, 591-598,601-606, 617-625, 633-643, 658-664, 676-682, 694-702, 710-719, 754-760,782-788, 802-808, 916-921, 942-948, 955-964, 973-979, 992-998,1006-1011, 1016-1023, 1030-1038, 1046-1053, 1059-1066, 1088-1098,1119-1126, 1129-1135, 1156-1171, 1173-1181, 1202-1210, 1255-1261,1268-1280, 1295-1310, 1312-1320, 1375-1381, 1406-1417, 1450-1471,1478-1492, 1498-1506, 1569-1578, 1603-1608, 1611-1624, 1648-1655,1663-1670, 1680-1698, 1702-1707, 1713-1719, 1737-1742, 1747-1753,1762-1769, 1771-1785, 1790-1804, 1811-1818, 1830-1836, 1838-1852,1874-1886, 1893-1899, 1902-1909, 1942-1948, 1952-1962, 1980-1986,2001-2017, 2020-2028, 2042-2050, 2052-2068, 2074-2079, 2083-2095,2107-2113, 2147-2155, 2177-2194, 2203-2211, 2236-2241, 2251-2258,2267-2274, 2285-2292, 2314-2328, 2330-2340, 2358-2365, 2390-2401,2408-2418, 2432-2453, 2463-2476, 2486-2507, 2528-2537, 2540-2548,2552-2558, 2568-2576, 2596-2601, 2610-2622, 2629-2638, 2653-2669,2718-2727, 2749-2767, 2777-2784, 2789-2795, 2806-2815, 2817-2824,2835-2843, 2847-2854, 2860-2881, 511-523, 612-630 and 1790-1803 of SEQID NO:209; 4-54, 61-68, 72-82, 86-93, 100-108, 115-130, 147-154,187-194, 196-207, 224-229, 236-251, 275-287 and 96-109 of SEQ ID NO:210;31-39, 62-69, 91-101, 158-172, 175-180, 186-193, 201-208, 210-223,243-250, 273-286, 293-299, 319-325, 343-354, 356-365, 368-384, 414-435,471-491, 512-518, 550-556, 567-581, 584-589, 633-639, 680-692, 697-708,716-721, 747-754, 779-786, 810-816 and 366-503 of SEQ ID NO:211; 5-20,22-48, 57-65, 96-101, 111-122, 130-145, 154-164, 170-181, 193-199,201-216, 224-241, 244-262, 281-323, 342-351, 359-367, 369-396, 406-416,424-433, 450-456, 485-491, 493-499, 501-515, 517-535 and 289-305 of SEQID NO:212; 4-17, 22-44, 53-60, 66-83, 87-94, 101-106, 110-116, 131-137,148-183, 189-207, 209-215, 233-242, 251-262, 264-272, 290-296, 308-327,359-373, 375-380, 397-405, 415-420, 426-433, 444-475, 478-484, 529-536,548-558 and 106-126 of SEQ ID NO:213; 4-38, 42-50, 58-64, 72-81, 92-118,140-146, 157-165, 172-192, 198-204, 208-216, 227-234, 238-258, 271-278,288-293, 311-322, 327-346, 357-370, 375-383, 395-409, 411-417, 425-432,436-445, 109-129 and 370-380 of SEQ ID NO:214; 23-30, 36-49, 52-64,86-94, 97-104, 121-129, 257-272, 279-286, 288-294, 307-327, 334-340,369-375, 377-386, 406-412, 418-423, 430-438, 441-447, 459-465, 469-476,482-488, 510-546, 550-580, 584-622, 638-645, 653-659, 675-683, 692-705,723-731, 752-761, 788-795 and 54-72 of SEQ ID NO:215; 11-33, 36-46,88-104, 116-126, 134-170, 189-195, 199-217, 225-250, 255-261, 266-273,280-291, 296-313, 334-341, 343-349, 354-360, 362-369, 373-380, 387-401,406-420 and 259-273 of SEQ ID NO:216; 9-14, 28-44, 57-64, 72-79, 86-93,104-111, 116-126, 142-150, 159-164 and 61-86 of SEQ ID NO:217; 10-17,26-33, 43-61, 69-95, 101-107, 109-125, 129-135, 137-144, 147-153,158-169, 177-187, 209-219, 221-232, 235-247, 261-268, 271-282, 296-302,306-347, 355-362, 364-379, 386-399, 409-418, 424-442, 451-460, 467-479,490-498 and 60-74 of SEQ ID NO:218; 8-14, 20-31, 65-84, 94-99, 154-179,193-207, 238-253 and 96-118 of SEQ ID NO:219; 4-24, 30-44, 47-62, 84-93,108-116, 124-133, 136-141, 201-209, 217-223, 228-235, 238-245, 247-270,275-285, 290-314, 328-338, 342-349, 353-365, 375-383, 386-392, 394-402,417-427, 443-459, 465-481, 492-514, 516-524, 550-566, 602-617, 630-639,666-676, 687-693, 719-730, 747-753, 783-790, 799-816, 824-831, 837-842and 167-189 of SEQ ID NO:220; 6-15, 18-28, 58-66, 84-101, 106-129,136-151, 154-165, 182-203, 205-211, 214-220, 222-228, 233-240, 251-260,270-277, 284-291, 306-315, 322-328, 363-369, 378-388, 392-405, 443-452,495-501, 512-523, 574-583 and 362-375 of SEQ ID NO:221; 5-25, 27-34,47-59, 64-70, 76-86, 145-158, 166-183, 189-202, 217-231, 235-242,260-270, 278-309 and 1-102 of SEQ ID NO:222; 4-19, 24-76, 78-83, 90-99,102-109, 114-122, 137-147, 154-174, 177-188, 203-212, 217-223, 227-239and 226-325 of SEQ ID NO:223; 7-37, 71-90, 94-109, 117-128, 141-153,179-192, 199-206, 225-231, 237-243, 258-264 and 40-51 of SEQ ID NO:224;13-19, 25-30, 46-59, 75-91, 101-107, 114-124, 129-135, 137-145, 160-167,171-179, 187-194, 209-215, 217-222, 229-239, 243-249, 257-265, 269-275,299-308, 310-327 and 282-300 of SEQ ID NO:225; 86-100, 216-230, 342-369,382-388, 424-430, 438-445, 452-458, 488-494, 501-518, 554-560, 568-574,584-592, 603-609, 611-629, 639-645, 652-661, 669-699, 708-714, 726-738,747-753, 763-775, 785-791, 794-807, 815-824, 826-845, 854-860, 863-868,870-883, 892-898, 901-906, 909-921, 930-937, 946-959, 968-974, 977-990,998-1007, 1009-1027, 1037-1043, 1046-1051, 1053-1066, 1075-1081,1084-1089, 1092-1103, 1113-1119, 1122-1135, 1143-1152, 1154-1172,1182-1188, 1191-1196, 1200-1210, 1220-1226, 1229-1235, 1237-1249,1259-1265, 1268-1281, 1289-1298, 1305-1318, 1328-1334, 1337-1343,1345-1357, 1367-1373, 1390-1396, 1405-1411, 1418-1423, 1426-1435,1445-1455, 1474-1483, 1493-1500, 1505-1512, 1517-1524, 1538-1544,1568-1578, 1595-1601, 1674-1682, 1687-1720, 1728-1736, 1738-1744,1754-1761, 1764-1774, 1798-1824, 1836-1842, 1886-1893, 1895-1903,366-781, 782-1518 and 1731-1747 of SEQ ID NO:226; 4-17, 20-39, 46-55,60-66, 102-110, 114-122, 125-131, 161-167, 172-178, 185-190, 195-202,218-232, 236-252, 264-291, 293-302, 309-315, 324-339 and 169-381 of SEQID NO:227; 5-10, 13-40, 42-53, 69-75, 83-89, 120-135, 150-161, 174-190,203-225, 229-247, 257-287, 318-348 and 30-200 of SEQ ID NO:228; 7-19,43-53, 64-72, 124-139, 52-84 and 120-131 of SEQ ID NO:229; 12-19, 39-48,58-100, 117-123, 154-162, 164-187, 189-195, 202-216, 218-235, 241-246,262-278, 315-328, 333-347, 354-366, 372-379, 391-405, 422-429, 431-442,444-450, 458-466, 478-485, 494-501, 504-510, 520-535, 573-580, 589-598,615-625, 666-676, 686-698, 722-729, 737-746, 756-767, 787-796, 805-816,824-829, 833-848, 856-864, 866-876, 879-886, 898-904, 918-924, 927-934,941-960, 967-978 and 561-575 of SEQ ID NO:230; 11-29, 49-55, 70-77,84-100, 102-112, 148-155, 160-177, 181-204 and 1-104 of SEQ ID NO:231;27-44, 64-71, 122-133, 151-156, 164-178, 214-220, 226-232, 235-244,253-262, 282-288, 294-310, 317-325, 350-356, 362-368, 376-383, 438-443,449-454, 459-464, 492-498, 500-511, 529-535, 538-546, 567-573, 597-603,660-665, 674-679, 724-734, 763-769, 773-784, 791-801, 807-815, 821-826,840-848, 863-868, 897-902, 908-928, 932-953, 956-975, 980-987, 990-996,1012-1018, 1042-1063, 1095-1116, 1149-1157, 1160-1167, 110-357, 358-501and 502-1161 of SEQ ID NO:232; 4-21, 64-71, 73-84, 128-138, 144-162,203-217, 240-263, 288-298, 300-308, 310-317, 325-351, 369-380, 391-411and 330-345 of SEQ ID NO:233; 5-11, 25-31, 39-48, 51-79, 89-98, 100-122,135-148, 166-201, 203-227, 230-250, 254-260, 266-272, 274-282, 299-305,328-337 and 31-45 of SEQ ID NO:234; 12-23, 29-48, 51-60, 66-72, 75-81,83-93, 103-115, 133-148, 168-174, 195-204, 222-229, 231-240, 242-251,270-280, 286-305, 322-344, 349-360, 364-370, 378-400, 421-441, 448-484,486-493, 495-501, 504-534, 547-561, 567-590, 597-607, 621-635, 643-649,658-685, 688-694, 702-711, 717-731, 737-742, 759-765, 767-772, 776-786,803-809, 815-825, 854-908, 910-919, 923-930, 942-948, 961-975, 994-1014and 915-940 of SEQ ID NO:235; 4-9, 32-47, 51-61, 75-96, 139-191 and1-124 of SEQ ID NO:236; 4-13, 17-38, 43-49, 55-76, 88-95, 110-121,128-146, 151-157, 162-214, 222-240, 243-249, 251-273, 275-281, 292-298,300-309, 312-320, 322-331, 355-369, 376-408, 446-460, 471-482, 485-509and 191-203 of SEQ ID NO:237; 4-21, 72-82, 89-103, 106-115, 118-124,140-146, 174-184, 191-200, 204-213, 218-224, 261-266, 282-293, 299-309,311-340, 342-358, 362-372, 381-389, 391-402, 413-421, 438-447, 457-464,470-478, 501-507, 545-560, 578-624, 631-641, 658-670, 680-689, 717-738,753-759, 795-805, 816-822, 830-838, 842-848, 869-881, 892-898, 33-51 and818-835 of SEQ ID NO:238; 4-21, 79-85, 156-177, 183-188, 206-214,243-249, 261-269, 287-292, 315-322, 334-345, 360-366, 374-390, 402-411,37-97 and 260-399 of SEQ ID NO:239; 4-9, 19-54, 58-78, 97-104, 111-120,126-134, 137-145, 163-173, 178-188, 193-203, 211-224, 246-286, 288-324,337-346, 355-362, 374-390, 392-398, 409-417 and 240-249 of SEQ IDNO:240; 5-12, 14-31, 35-41, 43-61, 82-92, 97-105, 134-145, 155-166,184-203, 215-223, 225-251, 272-279, 281-306, 310-345, 358-418, 435-473,482-490, 525-532, 538-547, 549-563, 578-604, 613-639 and 144-154 of SEQID NO:241; 53-59, 64-72, 74-100, 133-152, 154-172, 176-181, 207-214,225-238, 275-297, 304-310, 331-340, 362-367, 384-395, 403-410, 437-443,448-456, 482-490, 579-597, 602-610, 625-630, 633-651, 699-707, 709-715,734-743, 750-762 and 544-685 of SEQ ID NO:242; 12-18, 22-40, 45-83,89-97, 103-109, 147-153, 159-173, 195-204, 210-219, 243-253, 259-265,273-282, 303-309, 315-325, 332-340, 346-358, 362-367, 377-390, 393-402,418-426, 447-455, 467-480, 505-512, 514-525, 548-561, 566-576, 584-596,619-626, 638-645, 649-659, 661-680, 699-708, 714-720, 753-759, 766-772,775-781, 801-808, 202-218, 282-299, 339-350 and 617-628 of SEQ IDNO:243; 5-33, 52-62, 87-101, 111-135, 137-143, 145-152, 190-202,209-221, 233-245, 253-270 and 151-215 of SEQ ID NO:244; 19-29, 32-39,42-48, 75-94, 124-135, 137-145, 152-160, 176-182, 193-203, 215-236,266-273, 275-291, 297-306, 311-319, 322-342, 348-360, 369-378, 394-401and 48-64 of SEQ ID NO:245; 4-11, 13-33, 36-43, 53-63, 65-80, 112-129,134-141, 143-155, 157-168, 178-188, 191-199, 201-207, 215-229, 242-255,263-270, 283-315, 320-329, 333-338, 340-349, 412-426, 465-478, 485-490,498-512, 540-554 and 390-516 of SEQ ID NO:246; 4-18, 23-32, 41-47,54-70, 88-99, 104-111, 118-138, 143-148, 150-162, 168-175, 181-188,203-211, 214-220, 227-245, 251-268, 275-281, 287-296, 323-333 and 1-90of SEQ ID NO:247; 8-34, 38-49, 72-83, 85-91, 94-104, 112-125, 134-142,148-168, 181-189, 191-198, 202-214, 222-233, 242-254, 256-262, 273-278,287-294, 314-325 and 141-159 of SEQ ID NO:248; 4-24, 30-36, 47-75,82-105, 124-134, 151-157, 192-202, 208-214, 219-226, 234-247, 285-290,318-324, 332-340, 343-349, 380-386, 453-462, 472-478, 484-501, 531-540,550-557, 604-612, 620-625, 642-648, 652-671, 64-84, 93-180 and 181-446of SEQ ID NO:249; 12-18, 24-32, 68-75, 77-83, 96-101, 109-116, 129-136,152-164, 175-184, 190-199, 206-215, 224-233, 241-250, 258-264, 273-292,302-312, 319-331, 334-346, 348-368, 387-395, 408-416, 420-429, 437-452and 364-374 of SEQ if D NO:250; 11-28, 36-52, 60-67, 74-79, 108-116 and61-76 of SEQ ID NO:251; 20-27, 38-49, 69-74, 84-107, 138-145, 161-168,179-195, 210-226, 228-252, 267-281, 283-296, 305-311, 333-340, 342-356,361-372, 380-399, 401-414, 458-466, 475-481, 492-507, 515-520 and146-160 of SEQ ID NO:252; 43-61, 68-74, 76-90, 120-128, 130-149,156-161, 164-182, 206-234, 242-252, 269-274, 291-304, 332-345, 349-355,360-371, 374-388, 434-440, 447-453, 459-465, 469-496, 504-522 and261-285 of SEQ ID NO:253; 4-17, 24-30, 37-49, 87-98, 118-124, 126-136,144-171, 176-188, 206-214, 216-228, 233-240, 246-252, 262-271, 277-297,307-330, 333-342, 346-352, 355-361, 368-386, 391-400, 413-420, 474-480and 401-427 of SEQ ID NO:254; 15-26, 31-46, 51-72, 80-93, 96-109,131-137, 150-158, 179-185, 189-209, 211-219, 221-234, 241-247, 255-262,265-271, 283-288 and 173-190 of SEQ ID NO:255; 28-37, 39-45, 51-58,77-84, 89-97, 132-148, 171-180, 199-205, 212-218, 220-226, 257-265,273-300, 307-327, 334-340, 344-365, 385-390, 402-408, 426-436, 450-468,476-485 and 425-497 of SEQ ID NO:256; 4-25, 70-76, 80-88, 90-100,120-128, 162-169, 183-203, 261-277, 279-289, 291-297, 302-308, 321-327,339-353, 358-377, 392-401, 404-410, 414-422, 443-450, 456-461, 470-488,490-497, 510-535, 570-611, 618-630, 639-647, 649-660, 668-690, 702-716,718-724, 737-747, 750-764 and 497-509 of SEQ ID NO:257; 12-48, 50-64,99-108, 216-223, 235-241, 244-254, 262-274, 287-293, 310-316, 320-326,361-366, 377-383, 390-395, 408-414, 418-425, 438-444, 462-469, 494-505,524-530, 536-547, 551-566, 592-598, 601-613, 678-685, 687-695, 709-717,727-737, 751-757, 760-765, 772-778, 782-788, 801-807, 822-830, 859-868,870-878, 884-890, 898-903, 909-919, 953-969, 973-980, 990-1000,1002-1019, 1041-1047, 1059-1065, 1090-1095, 1116-1127, 1130-1139,1143-1149, 1151-1168, 1178-1183, 1188-1195, 1197-1209, 1213-1220,1226-1234, 1236-1247, 1255-1274, 1276-1282, 76-100, 270-284, 309-438,493-505, 786-942 and 947-967 of SEQ ID NO:258; 4-9, 24-34, 46-95,97-109, 119-130 and 138-156 of SEQ ID NO:259; 9-26, 28-35, 43-53, 55-68,83-92, 99-105, 110-135, 139-149, 157-162, 164-170, 173-183, 193-208,210-230, 239-245, 253-259, 263-271, 293-305, 310-320, 322-331, 336-343,351-364, 367-376, 92-107 and 154-173 of SEQ ID NO:260; 19-39, 52-62,108-117, 145-152, 160-168, 194-203, 229-240, 252-268, 280-287, 308-316,333-339, 383-390, 403-412, 414-424, 438-445, 464-472, 479-484, 489-505,510-526 and 247-260 of SEQ ID NO:261; 5-17, 25-52, 60-77, 105-113,118-125, 162-167, 228-234, 272-279, 328-334, 341-357, 381-395, 400-406,512-518, 557-569, 586-592, 645-651, 690-695, 701-709, 720-726, 733-743,751-758, 781-786, 879-886, 929-934, 939-944, 952-960, 965-975, 994-1001,1039-1045, 1102-1109, 1164-1181, 1198-1206, 1223-1229, 1253-1259,1283-1292, 1312-1317, 1339-1349, 1360-1370, 1389-1398, 1400-1412,1452-1465, 1470-1484, 1490-1497, 1519-1525, 1554-1564, 1578-1591,1623-1636, 1638-1646, 1669-1679, 1685-1697, 1704-1711, 1713-1720,1730-1736, 1738-1749, 1756-1764, 1778-1786, 1796-1803, 1817-1826,1849-1866, 1975-1993, 2017-2032, 2044-2053, 2070-2086, 2091-2109,2116-2127, 2156-2167, 2182-2188, 2197-2202, 2244-2252, 2281-2287,2290-2307, 2350-2361, 2383-2404, 2425-2433, 2445-2455, 2495-2505 and394-549 of SEQ ID NO:262; 9-24, 31-53, 57-67, 69-79, 84-114, 133-141,144-172, 178-186 and 13-46 of SEQ ID NO:263; 4-25, 27-35, 43-52, 59-70,79-91, 115-130, 136-152, 154-163, 170-179 and 1-58 of SEQ ID NO:264;4-30, 49-55, 71-80, 96-105, 111-126, 139-146, 149-162, 239-245, 279-285,290-296, 300-307, 331-337, 343-350 and 250-351 of SEQ ID NO:265; 9-27,34-41, 43-51, 92-111, 114-120, 123-131, 139-150, 156-171, 176-186,188-204, 229-241, 252-258, 266-279, 288-297, 319-334, 338-348, 373-379,389-398, 431-439, 479-484 and 214-398 of SEQ ID NO:266; 4-15, 18-27,47-52, 68-83, 91-97, 104-110, 115-121, 139-147, 157-164, 198-206,227-236, 241-254, 264-273, 278-289, 311-320, 353-361, 372-383, 405-420,426-434 and 232-386 of SEQ ID NO:267; 4-10, 24-34, 91-97, 129-141,156-163, 184-190, 205-219, 229-235, 256-273, 278-285 and 93-116 of SEQID NO:268; 7-29, 35-54, 71-83, 85-91, 104-111, 122-134, 138-144,146-154, 158-174, 177-183, 186-201, 207-215, 223-235, 240-247, 262-273,275-283, 287-292 and 48-66 of SEQ ID NO:269; 7-27, 31-47, 49-70, 75-102,110-149, 157-171, 217-223, 235-251, 294-302, 358-364, 367-375, 387-393,395-412, 423-430, 441-451, 456-470, 472-486, 488-495, 499-509, 515-529,536-549, 556-570, 574-603, 607-615, 625-633, 642-658, 670-676, 683-702,708-716, 720-726, 747-756, 763-784, 803-812, 815-826 and 475-490 of SEQID NO:270; 7-22, 30-38, 53-59, 64-75, 83-95, 97-112, 120-131, 133-142,145-151, 154-166, 172-180, 189-203, 227-238, 277-287, 9-156 and 174-287of SEQ ID NO:271; 13-23, 25-32, 111-117, 150-164, 185-193, 207-212,216-224, 230-236, 263-272, 304-311, 342-348, 374-385, 391-407, 444-458,480-487, 489-499, 523-542, 544-558, 572-579, 620-640, 686-696, 703-710,742-755, 765-772, 817-822, 830-837, 865-872, 931-937 and 66-86 of SEQ IDNO:272; 4-27, 49-56, 62-70, 86-92, 121-127, 151-163, 170-182, 195-202,212-226, 237-243 and 234-254 of SEQ ID NO:273; 4-10, 13-24, 39-51,62-78, 92-104, 107-117, 134-141, 156-161, 166-181, 210-216, 222-229,256-266, 273-280, 297-304, 313-330, 336-349, 371-376, 433-439, 443-448,488-493, 506-515, 527-534, 560-572, 575-583, 587-593 and 252-483 of SEQID NO:274; 4-15, 21-38, 45-56, 81-95, 102-108, 118-130, 133-147,152-162, 166-171, 199-204, 211-218, 230-240, 253-261, 274-283, 288-294,312-317, 325-336, 344-357, 391-414 and 24-146 of SEQ ID NO:275; 26-31,38-56, 65-82, 90-101, 112-119, 123-153, 175-188, 197-216, 234-242,249-265, 273-286, 290-305, 327-335, 338-346, 361-372, 394-404 and290-306 of SEQ ID NO:276; 17-26, 43-48, 50-73, 81-93, 95-107, 139-146,158-168, 171-176, 190-196, 202-212, 216-223, 243-266, 274-282, 308-313,324-330, 344-378, 380-387, 403-422, 427-443, 448-455, 457-465, 491-515,517-528, 553-567, 589-599, 610-617, 642-648, 670-697, 709-717, 726-743,745-759, 769-803, 807-823, 840-849 and 820-851 of SEQ ID NO:277; 4-18,39-48, 53-63, 66-90, 102-117, 125-134, 137-145, 156-162, 169-197, 26-40and 56-80 of SEQ ID NO:278; 21-33, 36-42, 49-60, 68-76, 91-105, 123-130,141-161, 169-178, 185-190, 192-199, 205-214, 223-233, 239-247, 260-269,284-293, 300-314, 324-352, 357-364, 373-382, 389-403, 420-432, 438-446,466-471, 477-484, 503-509, 549-556, 558-576, 600-623, 625-635, 654-661,663-669, 671-687, 702-716, 735-741, 744-750, 757-766, 776-786, 807-815,824-832, 854-860, 863-897, 909-915, 920-946, 952-959, 982-997,1024-1038, 1049-1055, 1071-1085, 1104-1113, 1121-1132, 1138-1150,1187-1196, 1212-1221, 1227-1236, 1257-1262, 1264-1278, 1282-1294,1307-1318, 1353-1370, 1382-1388, 1396-1409, 1434-1440, 1446-1454,1465-1478, 1485-1513, 1516-1529, 1540-1545, 1563-1568, 1575-1593,1607-1616, 1628-1645, 1648-1661, 1676-1682, 1689-1697, 1713-1719,1739-1749, 1753-1758, 1763-1774, 1797-1803, 1807-1846, 1855-1874,1877-1891, 1893-1907, 1912-1925, 1931-1943, 1955-1965, 1976-1990,2032-2043, 2045-2051, 2099-2105, 2131-2138, 2161-2179, 2188-2199,2205-2216, 2219-2227, 2235-2245, 2247-2267, 2277-2288, 2294-2304,2314-2326, 2346-2358, 2365-2377, 2383-2402, 2407-2423, 2437-2450,2454-2473, 2489-2497, 2525-2531, 2557-2570, 2580-2587, 2589-2599,2621-2641, 2647-2653, 2661-2677, 2685-2690, 2697-2717, 2722-2733,2739-2777, 2786-2793, 2801-2808, 2811-2822, 2825-2835, 2838-2845,2859-2871, 2877-2883, 213-344, 954-1080 and 2524-2733 of SEQ ID NO:279;10-16, 18-23, 28-41, 63-69, 77-91, 101-109, 118-136, 146-153, 155-162,168-179, 192-207, 217-226, 229-235, 239-254, 279-286, 294-307, 313-319,334-341, 344-353, 363-377, 390-396 and 178-328 of SEQ ID NO:280; 18-42,68-84, 89-95, 100-105, 107-115, 125-135, 154-177, 189-195, 205-228,236-243, 252-259, 279-300, 309-316, 323-331, 340-351, 353-364, 377-402and 85-97 of SEQ ID NO:281; 4-18, 26-32, 66-76, 100-126, 151-159,178-186, 188-194, 200-210, 241-248, 253-259, 262-279, 284-291, 307-313,315-322, 327-337, 376-386, 399-407, 432-441, 467-473, 487-497, 499-505,543-549, 560-568, 585-593, 598-604, 608-614, 630-642, 647-653, 690-703,717-730, 21-200 and 468-480 of SEQ ID NO:282; 17-49, 52-58, 62-73,78-97, 100-117, 122-172, 185-190, 193-217, 225-236 and 33-42 of SEQ IDNO:283; 7-39, 50-58, 73-89, 96-107, 109-120, 126-142, 152-170, 178-202,205-211, 224-244, 249-259, 261-270, 300-310, 312-325 and 158-169 of SEQID NO:284; 4-31, 40-64, 71-82, 85-92, 102-124, 126-139, 147-152,159-173, 176-188, 195-207, 210-216, 234-241, 249-256, 258-276, 279-293,296-302, 310-315, 349-356, 363-378, 380-403, 411-426, 435-441, 448-459,463-476, 488-494 and 201-221 of SEQ ID NO:285; 5-13, 15-74, 87-104,107-120, 123-129, 136-145, 150-191, 193-206, 227-248, 250-264, 278-302,304-323, 332-378, 384-407, 409-419, 425-457, 462-471, 474-497, 511-545,555-564, 571-578, 585-598, 640-647, 669-675, 682-691, 693-705, 729-743,752-761, 772-780, 786-804, 808-818, 822-846, 858-880, 884-900, 910-939,941-947, 962-971, 973-988, 998-1003, 1007-1027 and 236-259 of SEQ IDNO:286; 4-19, 27-68, 81-111, 121-160 and 60-79 of SEQ ID NO:287; 4-37,40-46, 52-57, 199-205, 222-229, 236-244, 250-267, 269-282 and 27-197 ofSEQ ID NO:288; 4-16, 24-30, 32-38, 63-75, 86-92, 98-111, 113-126,160-165, 170-180, 198-204, 227-233, 239-245, 253-273, 308-314, 352-365,382-387, 395-403, 423-429, 472-482, 484-493, 501-507, 518-526, 536-541,543-550, 556-562, 586-600, 626-633, 649-661, 680-688 and 546-559 of SEQID NO:289; 16-33, 48-59, 63-71, 77-92, 94-109, 117-124, 139-151,169-181, 184-227, 233-249, 251-261, 263-275, 282-294, 297-321, 326-332,341-355, 383-399 and 258-272 of SEQ ID NO:290; 11-26, 31-39, 43-52,55-62, 64-70, 80-94, 123-133, 135-141, 172-181, 185-206, 209-218,224-230, 238-244, 251-262, 264-271, 290-301, 306-324, 333-340, 350-357,367-375, 390-397, 434-441, 443-448, 77-226 and 350-429 of SEQ ID NO:291;4-13, 22-27, 31-45, 50-59, 72-96, 99-114, 131-141, 143-150, 159-176,180-186, 189-198, 208-214, 234-253, 271-287, 294-299, 310-366, 382-390,398-416, 424-443 and 283-305 of SEQ ID NO:292; 9-26, 30-53, 62-72,86-95, 112-122, 136-145, 153-160, 209-221, 227-237, 241-268, 281-288,291-298, 308-314, 321-328, 336-346, 351-379, 388-397, 409-416, 423-433,443-481, 511-519 and 213-232 of SEQ ID NO:293; 12-18, 25-31, 38-50,59-67, 71-82, 96-126 and 76-88 of SEQ ED NO:294; 4-25, 39-44, 64-71,74-88, 100-113, 128-138, 151-162, 164-177, 185-190, 204-213, 233-239,246-254, 281-286, 293-306, 309-318, 333-347, 349-359, 385-398, 404-423,458-465, 477-484, 490-499, 501-533, 554-566, 582-590, 596-616, 624-629,631-639, 654-680, 694-720, 735-743 and 2-100 of SEQ ID NO:295; 4-16,36-41, 52-75, 98-107, 109-117, 122-128, 133-139, 141-155, 159-165,169-182, 187-193, 195-201, 211-224, 230-236, 247-269, 278-290 and 75-92of SEQ ID NO:296; 7-21, 25-33, 37-43, 87-94, 103-120, 131-147, 168-174,197-203, 207-212, 227-237, 247-257, 263-271, 279-287, 298-306, 320-325,332-340, 363-374, 379-384, 390-401, 403-414, 428-433, 448-457, 462-475,483-490, 513-519, 525-535, 543-554, 559-566, 571-620, 625-631, 636-642,659-670, 688-706, 708-723, 770-779, 787-793, 796-807, 820-840, 848-854,863-874, 895-905, 912-919, 934-942, 968-975, 983-1000, 1012-1019,1026-1036, 1050-1060, 1064-1070, 1081-1091, 1094-1108, 1112-1118,1140-1152, 1164-1169, 1172-1180, 1187-1192 and 732-748 of SEQ ID NO:297;23-40, 42-59, 66-73, 78-97, 111-128, 130-141, 157-166, 178-183 and 53-71of SEQ ID NO:298; 4-27, 38-44, 47-57, 59-85, 99-106, 114-121, 154-166,181-186, 193-198, 238-244, 253-262, 272-278, 287-299, 314-320, 338-350,358-368, 382-388, 407-416, 433-446, 456-461, 463-473 and 86-195 of SEQID NO:299; 5-24, 38-59, 64-80, 87-99, 105-126, 134-142, 149-163,165-179, 181-202, 205-220, 227-233, 243-250, 257-263 and 87-245 of SEQID NO:300; 5-32, 47-53, 66-79, 81-97, 115-151, 155-174, 183-188,196-210, 215-226, 230-238, 253-258, 263-270, 276-282, 295-301, 304-325,334-344, 360-390, 397-412, 425-432, 434-462, 478-494, 508-526, 539-564,571-579, 347-371 and 375-386 of SEQ ID NO:301; 4-15, 36-44, 49-56,60-66, 68-82, 84-103, 109-115, 118-141, 147-154, 160-168, 176-185 and26-39 of SEQ ID NO:302; 7-13, 23-33 and 13-21 of SEQ ID NO:303; 2-10 ofSEQ ID NO:304; 4-9, 12-18, 35-42, 49-62 and 6-18 of SEQ ID NO:305; 19-25and 1-13 of SEQ ID NO:306; 15-21, 27-45 and 12-25 of SEQ ID NO:307;14-20 and 1-14 of SEQ ID NO:308; 4-18 and 13-26 of SEQ ID NO:309; 8-21and 2-20 of SEQ ID NO:310; 4-14 and 4-16 of SEQ ID NO:311; 3-12 of SEQID NO:312; 6-14, 6-25, 35-57 and 2-14 of SEQ ID NO:313; 6-25, 35-57 and17-31 of SEQ ID NO:314; 14-25, 32-46 and 5-19 of SEQ ID NO:315; 18-31and 5-16 of SEQ ID NO:316; 19-24 and 4-26 of SEQ ID NO:317; 13-21,29-34, 47-58, 61-73 and 36-47 of SEQ ID NO:318; 4-15 and 5-24 of SEQ IDNO:319; 6-18 of SEQ ID NO:320; 13-20 and 4-13 of SEQ ID NO:321; 15-23 ofSEQ ID NO:322; 4-9 and 7-21 of SEQ ID NO:323; 1-10 of SEQ ID NO:324;4-14 of SEQ ID NO:325; 4-17, 35-41, 46-89, 93-98 and 70-88 of SEQ IDNO:326; 1-13 of SEQ ID NO:327; 4-16, 26-32 and 25-38 of SEQ ID NO:328;8-15, 23-28 and 4-17 of SEQ ID NO:329; 4-12 and 1-15 of SEQ ID NO:330;4-29, 31-42, 52-58 and 6-16 of SEQ ID NO:331; 4-9, 24-32 and 9-19 of SEQID NO:332; 4-12, 18-27 and 5-18 of SEQ ID NO:333; 4-11, 37-56, 58-92 and18-29 of SEQ ID NO:334; 8-28 and 20-35 of SEQ ID NO:335; 4-15 of SEQ IDNO:336; 4-23, 27-39, 55-63 and 35-58 of SEQ ID NO:337; 6-26, 28-54 and28-47 of SEQ ID NO:338; 4-10, 38-52, 58-82 and 30-49 of SEQ ID NO:339;4-22, 29-35, 44-50, 53-68, 70-80 and 20-33 of SEQ ID NO:340; 22-28,30-36 and 18-33 of SEQ ID NO:341; 4-11, 13-21, 25-30 and 20-30 of SEQ IDNO:342; 10-22 and 10-23 of SEQ ID NO:343; 4-11 and 9-20 of SEQ IDNO:344; 14-25, 32-46 and 6-19 of SEQ ID NO:345; 5-30 and 14-33 of SEQ IDNO:346; 4-15, 28-35, 46-55, 59-65, 76-84 and 9-24 of SEQ ID NO:347;27-33 and 5-19 of SEQ ID NO:348; 5-13 and 8-18 of SEQ ID NO:349; 9-22,24-34 and 21-40 of SEQ ID NO:350; 4-17, 35-41, 46-89, 93-98 and 71-89 ofSEQ ID NO:351; 4-12, 14-24 and 2-17 of SEQ ID NO:352; 9-17 and 5-16 ofSEQ ID NO:353; 7-41, 48-58, 63-75, 80-89 and 43-53 of SEQ ID NO:354;4-22, 25-30 and 4-14 of SEQ ID NO:355; 4-55 and 18-33 of SEQ ID NO:356;262-280 of SEQ ID NO:179; 131-146 of SEQ ID NO:186; 207-224 of SEQ IDNO:188; 27-50, 203-217 and 313-325 of SEQ ID NO:189; 110-129 of SEQ IDNO:192; 156-179, 174-197, 192-215, 210-233, 228-251 and 246-267 of SEQID NO:190; 377-400 of SEQ ID NO:196; 34-43, 234-257 and 350-367 of SEQID NO:203; 304-327 of SEQ ID NO:207; 25-48, 43-66 and 61-82 of SEQ IDNO:222; 398-421, 416-439, 434-457, 452-475, 470-493, 488-511, 506-529,524-547, 621-644, 639-664, 707-730, 725-748, 743-766, 761-784, 779-802,797-820, 984-1007, 1002-1025, 1020-1043, 1038-1061, 1056-1079,1074-1097, 1092-1115, 1286-1309, 1304-1327, 1322-1345, 1340-1363,1358-1381, 1376-1399, 1394-1417, 1412-1435, 1430-1453, 1448-1471,1466-1489 and 1484-1507 of SEQ ID NO:226; 188-211, 206-229, 224-247,242-265, 260-283 and 278-296 of SEQ ID NO:227; 56-79 and 122-132 of SEQID NO:229; 35-46 of SEQ ID NO:231; 178-201, 196-219, 214-237, 232-255,250-273, 268-291, 379-402, 397-420, 415-438, 433-456, 451-474, 642-665,660-683, 678-701, 696-719, 714-737, 732-755, 750-773, 768-791, 899-922,917-940, 935-958, 1037-1060, 1055-1078, 1073-1096 and 1091-1114 of SEQID NO:232; 330-346 of SEQ ID NO:233; 571-594, 589-612, 607-630, 625-648,643-666 and 661-684 of SEQ ID NO:242; 188-207 of SEQ ID NO:244; 61-84,308-331, 326-349, 344-367, 362-385, 380-403 and 398-421 of SEQ IDNO:249; 79-98, 345-366, 844-867, 870-887 and 890-905 of SEQ ID NO:258;94-109 of SEQ ID NO:268; 188-207 of SEQ ID NO:272; 290-306 of SEQ IDNO:276; 826-849 of SEQ ID NO:277; 228-252, 247-270, 265-288, 283-306,301-324, 955-978, 973-996, 991-1014, 1009-1032, 1027-1050, 1045-1068,2533-2556, 2551-2574, 2569-2592, 2587-2610, 2605-2628 and 2623-2646 ofSEQ ID NO:279; 86-109 and 104-127 of SEQ ID NO:288; 546-560 of SEQ IDNO:289; 260-271 of SEQ ID NO:290; 106-129, 124-147, 142-165, 160-183,178-201 and 375-398 of SEQ ID NO:291; 284-307 of SEQ ID NO:292; 362-385of SEQ ID NO:301.
 41. The hyperimmune serum-reactive antigen or fragmentof claim 38, comprising at least 6 contiguous amino acids of any of SEQID NOs: 179-356.
 42. The hyperimmune serum-reactive antigen or fragmentof claim 38, comprising at least 8 contiguous amino acids of any of SEQID NOs: 179-356.
 43. The hyperimmune serum-reactive antigen or fragmentof claim 38, comprising at least 10 contiguous amino acids of any of SEQID NOs: 179-356.
 44. The hyperimmune serum-reactive antigen or fragmentof claim 38, further defined as directed against H. pylori.
 45. Apharmaceutical composition comprising a hyperimmune serum-reactiveantigen or fragment of claim
 38. 46. The pharmaceutical composition ofclaim 45, wherein the hyperimmune serum-reactive antigen or fragment isdirected against H. pylori.
 47. The pharmaceutical composition of claim45, further defined as comprising at least two different hyperimmuneserum-reactive antigens and/or fragments.
 48. The pharmaceuticalcomposition of claim 47, wherein the at least two different hyperimmuneserum-reactive antigens and/or fragments are both directed against H.pylori.
 49. The pharmaceutical composition of claim 45, furthercomprising an immunostimulatory substance.
 50. The pharmaceuticalcomposition of claim 49, wherein the immunostimulatory substance is apolycationic polymer, an immunostimulatory deoxynucleotide (ODN), apeptide containing at least two LysLeuLys motifs, a neuroactivecompound, alum, or a Freund's complete or incomplete adjuvant.
 51. Thepharmaceutical composition of claim 50, wherein the polycationic polymeris a polycationic peptide.
 52. The pharmaceutical composition of claim50, wherein the neuroactive compound is human growth hormone.
 53. Thepharmaceutical composition of claim 45, further defined as a vaccine.54. The pharmaceutical composition of claim 53, further defined as avaccine for treatment and/or prevention of H. pylori infection.
 55. Amethod of vaccinating a subject comprising: obtaining a pharmaceuticalcomposition of claim 45; and administering the pharmaceuticalcomposition to a subject; wherein the subject is vaccinated.
 56. Themethod of claim 55, wherein the subject is a human.
 57. The method ofclaim 55, further defined as a method of treating and/or preventing H.pylori infection in the subject.
 58. The method of claim 55, wherein thehyperimmune serum-reactive antigen or fragment is directed against H.pylori.
 59. The method of claim 55, wherein the pharmaceuticalcomposition comprises at least two different hyperimmune serum-reactiveantigens or fragments thereof.
 60. The method of claim 59, wherein theat least two different hyperimmune serum-reactive antigens or fragmentsthereof are both directed against H. pylori.